CN104540949A

CN104540949A - Methods for stabilizing production of acetyl-coenzyme a derived compounds

Info

Publication number: CN104540949A
Application number: CN201380041771.9A
Authority: CN
Inventors: 江汉笑; A·梅多斯
Original assignee: Total Marketing Services SA; Amyris Inc
Current assignee: TotalEnergies Marketing Services SA; Amyris Inc
Priority date: 2012-08-07
Filing date: 2013-08-07
Publication date: 2015-04-22
Anticipated expiration: 2033-08-07
Also published as: AU2013299608A1; US10927382B2; US20210155939A1; CA2879178C; BR112015002724A2; BR112015002724B1; EP2882856B1; US20150299713A1; EP2882856A1; AU2013299608B2; WO2014025941A1; CN104540949B; CA2879178A1

Abstract

The present disclosure relates to the use of a switch for the production of heterologous non-catabolic compounds in microbial host cells. In one aspect, provided herein are genetically modified microorganisms that produce non-catabolic compounds more stably when serially cultured under aerobic conditions followed by microaerobic conditions, and methods of producing non-catabolic compounds by culturing the genetically modified microbes under such culture conditions. In another aspect, provided herein are genetically modified microorganisms that produce non-catabolic compounds more stably when serially cultured in the presence of maltose followed by the reduction or absence of maltose, and methods of producing non-catabolic compounds by culturing the genetically modified microbes under such culture conditions.

Description

For the method that stable acetyl-coenzyme A derivative compound generates

Technical field

The disclosure relates to the purposes that oxygen responsiveness promotor generates for the non-alienation compound of allos regulating and controlling genetically modified host cell as genetic switch.

Background of invention

The appearance of synthetic biology brings organism of fermentation and produces biofuel from renewable source, the prospect of chemical preparations and biomaterial with technical scale and quality.Such as, in microorganism host, successfully construct functional non-native organism approach, for the production of the precursor (see such as Martin et al., Nat Biotechnol 21:796-802 (2003)) of antimalarial artemisinin; Fatty acid derived fuel and chemical preparations (such as fatty ester, fatty alcohol and and wax; See such as Steen et al., Nature 463:559-562 (2010)); Form the polyketide synthase (see such as Ma et al., Science 326:589-592 (2009)) of pravastatin; With polyketone (see such as Kodumal, Proc Natl Acad Sci USA 101:15573-15578 (2004)).But whether the production cost that the business success of synthetic biology can depend on renewable product greatly can become the production cost being comparable to or can be compared advantageously with their corresponding non-renewable counterparts.

Strain stability can be that of industrial fermentation cost mainly drives item because its impact continuously ferment can the time span of production run.Strain stability refers generally to the ability that microorganism maintains the favourable production feature (i.e. high yield (g of compound every gram substrate) and productivity (gram often liter of fermention medium is per hour)) of non-alienation tunning in the incubation time extended.Especially, genetic stability (namely producing the appointment gene frequency generating relevant gene in micropopulation with product changes little of unconverted tendency in time) plays Main Function in lasting output of products.

For the non-alienation fermentation of the product (according to definition, this product consumes metabolizable energy and carbon, and they can be used for generating more cell originally) except biomass, instable mechanism is double: evolve and suddenly change and select.First, spontaneous and random production development loss mutation.Secondly, the growth velocity of the cell that product yield reduces or " suitability " advantage cause the low producer to sweep across final group, and reduce gross culture performance thus.This phenomenon can be called " strain degeneration ".

Brazil's alcohol fuel fermentation realizes obtaining the high productive rate of ethanol from sugar to long period section, namely theoretical maximum productive rate about 90%.This part is dissimilarity because of alcohol production: its generates the sugar that 2 ATP per molecules generate, and not relate to oxygen be exactly redox reaction balance.Be mutated into the cell not generating ethanol be less compliant in the hypoxia condition of fermentor tank under and this group can not be swept across.This allows industrial alcohol fermentation to run through Seasonal Cycle major part yeast biomass, makes sugar be transformed into yeast-cell biomass thus and minimizes and nearly all sugar is guided to alcohol production.This expansion breeding and re-using of biomass improve the efficiency of alcohol production: running expenditure reduces because each cycle period less sugar become biomass (i.e. productive rate raising); And fund expenditure reduces, because build inoculation biomass used to need less and less fermentor tank.

Comparatively speaking, being created on of many acetyl-CoA derived hydrocarbon (such as isoprenoid, lipid acid, and polyketone) is generally non-dissimilarity in nature; They require ATP usually, NADPH, and the clean input of carbon, usually supply the redoxomorphism that a large amount of oxygen helps equilibrium system.This type of environment makes towards more low-yielding product, and higher biomass generate genotypic evolution advantageously, and causes the strain degeneration of higher rate.

Reducing a kind of mode of negative selective pressure generating non-alienation product is close product formation during the period not wanting this product, such as must generate biomass with during making the maximized fermentation stage of fermentor tank productivity.So, this area needs during fermentation to control the switch that acetyl-CoA derivative compound generates opportunity.

Summary of the invention

Provide the zymotechnique for generating the non-alienation compound of allos from genetically modified host cell herein.In some embodiments, this technique comprises two stages: the construction phase, and period, non-alienation compound generated substantive reduction ("Off" stage), cellular biomass accumulation simultaneously; And the production phase, non-alienation compound generated and opened period.So, generate relevant negative selective pressure with non-alienation compound to alleviate during not needing the fermentation stage generated.What during the construction phase, non-alienation compound generated reduce or eliminate cause (i) construction phase during the growth velocity of cell raise; (ii) during production phase, the production stability of bacterial strain raises.It is more of a specified duration that this causes the generation of non-alienation compound to continue, and improves overall production rate and/or the productivity of bacterial strain thus.Favourable thing, the "Off" that in the fermentation process provided herein, non-alienation compound generates and "On" state are via what easily obtain, and affordable, the condition relevant with industry controls.

On the one hand, the "Off" that in fermentation culture, non-alienation compound generates and "On" state are by the oxygen level between yeast phase, such as, in the substratum amount of dissolved oxygen, is combined and drives the oxygen sensitivity promotor of the genetic expression realizing the path enzyme that the non-alienation compound of allos generates to control.These methods utilize the observations that can provide oxygen when culturing engineering is transformed into the cell generating the non-alienation compound of allos with finite quantity.These cells can maintain growth and viability under micro-aerobic condition, save the cost relevant with running complete aerobic fermentation process thus.In some embodiments, once host cell group reaches the density being enough to picture supply oxygen oxygen consumed so fast, just micro-aerobic condition can be realized.Advantageously, by by approach gene expression and the coupling of oxygen sensitivity promotor, compound generate only the oxygen consumption of host cell group is high must be enough in fermentor tank, realize micro-aerobic condition time (this is at the end of the construction phase, in other words, for high efficiency compound generate achieve optimum cell density time effectively occur) open.So, approach gene expression starts the tight coupling of desirable population density with realization to the production phase.Thus, the method provided herein utilizes oxygen level and genetic switch to realize "Off" and the "ON" stage of the improvement zymotechnique generated for the non-alienation compound of allos.

So, provide a kind of method for generating the non-alienation compound of allos in genetically modified host cell herein, the method comprises:

A () cultivates the genetically modified host cell of a group under aerobic condition in the substratum comprising carbon source, wherein this host cell comprises coding for generating one or more heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of this allos, wherein the expression of these one or more enzymes is just regulated by the activity of micro-aerobic responsiveness promotor, and wherein this aerobic condition limits the amount of the non-alienation compound of allos generated by this host cell; And

B () cultivates described group or its subgroup under micro-aerobic condition in the substratum comprising carbon source, the non-alienation compound that wherein said micro-aerobic condition improves described group or its subgroup generates.

In some embodiments, this micro-aerobic responsiveness promotor is saltant type DAN1 promotor.In some embodiments, this saltant type DAN1 promotor comprises the sequence being selected from lower group: SEQ ID NO:1,2,3,4,5,6,7,8,9 and 10.In some embodiments, this saltant type DAN1 promoter sequence comprises SEQ ID NO:1.In some embodiments, this saltant type DAN1 promoter sequence comprises SEQ ID NO:2.

In some embodiments, one or more heterologous nucleic acids of one or more enzymes of this micro-aerobic responsiveness promotor and this coding enzymatic route are operatively connected, and described micro-aerobic condition improves the expression of one or more enzymes of this enzymatic route.In some embodiments, the heterologous nucleic acids of this micro-aerobic responsiveness promotor and encoding transcription instrumentality is operatively connected, this transcriptional is just regulating the expression of one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route, and described micro-aerobic condition improve this transcriptional expression.In some embodiments, this transcriptional is Gal4p, and one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route are operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10.In some embodiments, this host cell comprises the functional destruction of Gal80p further.

In some embodiments, this micro-aerobic condition dissolved oxygen concentration comprised in this substratum is less than about 20%, is less than about 15%, is less than about 10%, or be less than about 5%.In some embodiments, this micro-aerobic condition dissolved oxygen concentration comprised in this substratum is about 0%.In some embodiments, this micro-aerobic condition causes the oxygen uptake rate of this host cell to be less than about 50 mmoles, is less than about 40 mmoles, is less than about 30 mmoles, is less than about 20 mmoles and often rises substratum, or is less than about 10 mmoles and often rises substratum.In some embodiments, this micro-aerobic condition causes the ratio oxygen uptake rate of this host cell to be less than about 30 mmoles, is less than about 25 mmoles, be less than about 20 mmoles, be less than about 15 mmoles, be less than about 10 mmoles, or it is per hour to be less than about 5 mmoles every gram of dry cell weight.

In some embodiments, the non-alienation Compound Phase of allos realized in the aerobic fermentation process do not limited by the activity of this micro-aerobic responsiveness promotor by the expression of the allos non-alienation compound generated in this group of genetically modified host cells are between the incubation period of step (b) and one or more enzymes in this enzymatic route is than improving.

Also provide a kind of method for generating heterologous isoprenoid in genetically modified host cell herein, the method comprises:

A () cultivates the genetically modified host cell of a group under aerobic condition in the substratum comprising carbon source, wherein this host cell comprises:

One or more heterologous nucleic acids of one or more enzymes of (i) coding mevalonic acid (MEV) approach, it is operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10; With

(ii) nucleic acid of coding Gal4p, itself and micro-aerobic responsiveness promotor are operatively connected;

Wherein this aerobic condition limits the amount of the heterologous isoprenoid generated by this host cell; And

B () cultivates described group or its subgroup under micro-aerobic condition in the substratum comprising carbon source, the heterologous isoprenoid that wherein said micro-aerobic condition improves described group or its subgroup generates.

On the other hand, the "Off" that non-alienation compound in fermentation culture generates and "On" state, by the amount of sugared maltose in substratum, are combined and regulate the maltose responsiveness promotor of the genetic expression realizing the path enzyme that the non-alienation compound of allos generates to control.Advantageously, by by approach gene expression and the coupling of maltose susceptibility promotor, can open or close compound by the amount controlling maltose in feed and generate.Such as, maltose responsiveness promotor can be routed to "ON" lock, generates for non-alienation compound inducing heterogenous under maltose existence.Or maltose responsiveness promotor can be routed to "Off" lock, under existing at maltose, the down regulator being used for the enzymatic route that compound generates is induced to express.Thus, the method provided herein utilizes the maltose level in substratum and genetic switch to realize "Off" and the "ON" stage of the improvement zymotechnique generated for the non-alienation compound of allos.

A () cultivates the genetically modified host cell of a group in the substratum comprising carbon source (comprising maltose), wherein this host cell comprises coding for generating one or more heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of this allos, wherein the expression of these one or more enzymes is by the active negative regulator of maltose responsiveness promotor, wherein there is the amount that maltose limits the non-alienation compound of allos generated by this host cell in this substratum; And

B () cultivates described group or its subgroup in the substratum comprising carbon source, wherein maltose lacks or is in enough low amount and makes this maltose responsiveness promotor no longer include activity, and the non-alienation compound of the allos of this host cell generates rising.

In some embodiments, the heterologous nucleic acids of this maltose responsiveness promotor and encoding transcription instrumentality is operatively connected, the expression of one or more heterologous nucleic acids of one or more enzymes of this this coding enzymatic route of transcriptional negative regulator, and the maltose in step (a) improves the expression of this transcriptional.In some embodiments, this transcriptional is Gal80p, this host cell comprises Gal4p further, and one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route are operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10.In some embodiments, this maltose responsiveness promotor comprises the sequence being selected from lower group: pMAL1 (SEQ ID NO:12), pMAL2 (SEQ ID NO:13), pMAL11 (SEQ ID NO:14), pMAL12 (SEQID NO:15), pMAL31 (SEQ ID NO:16) and pMAL32 (SEQ ID NO:17).In some embodiments, this maltose responsiveness promoter sequence comprises pMAL32 (SEQ ID NO:17).

In some embodiments, the substratum of step (a) comprises at least 0.1% (w/v) maltose.In some embodiments, the substratum of step (a) comprises 0.25% to 3% (w/v) maltose.In some embodiments, the substratum of step (b) comprises and is no more than 0.08% (w/v) maltose.In some embodiments, the non-alienation Compound Phase of allos realized in the zymotechnique that the expression of the allos non-alienation compound generated between the incubation period of step (b) by this group of genetically modified host cells and one or more enzymes of this enzymatic route does not limit by the activity of this maltose responsiveness promotor is than improving.

A () cultivates the genetically modified host cell of a group in the substratum comprising carbon source (comprising maltose), wherein this host cell comprises:

One or more heterologous nucleic acids of one or more enzymes of (i) coding mevalonic acid (MEV) approach, it is operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10;

(ii) nucleic acid of coding Gal4p; With

(iii) nucleic acid of coding Gal80p, itself and maltose responsiveness promotor are operatively connected;

Maltose wherein in this substratum limits the amount of the heterologous isoprenoid generated by this host cell; And

B () cultivates described group or its subgroup in the substratum comprising carbon source, wherein maltose lacks or is in enough low amount, makes this maltose responsiveness promotor no longer include activity, and the non-alienation compound of the allos of this host cell generates rising.

In some embodiments, the non-alienation compound of step (a) period of methods described herein generates 50,40,30,20 or 10% of the non-alienation compound generation being less than step (b) period.In some embodiments, the cultivation of step (a) is one section at least 12,24,36,48,60,72,84,96 or more than 96 hours.In some embodiments, the cultivation of step (a) reaches cell density (OD between 0.01 and 400 for being enough to make described group ₆₀₀) time period.In some embodiments, the cultivation of step (b) be one section 3 to 20 days.In some embodiments, this non-alienation compound generates is measure in productive rate (gram non-alienation compound every gram of carbon substrate generated) or productivity (it is per hour that gram non-alienation compound generated often rises substratum).In some embodiments, the method comprises this non-alienation compound of recovery further.

On the other hand, the fermenting composition generated by fermentation process described herein is provided herein.In some embodiments, this fermenting composition comprises the genetically modified host cell of a group in the substratum comprising carbon source, wherein this host cell comprises coding for generating one or more heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of this allos, and wherein the expression of these one or more enzymes is just regulated by the activity of micro-aerobic responsiveness promotor.In some embodiments, one or more heterologous nucleic acids of one or more enzymes of this micro-aerobic responsiveness promotor and this coding enzymatic route are operatively connected, and wherein the expression of one or more enzymes of this enzymatic route raises under micro-aerobic fermentation condition.In some embodiments, the heterologous nucleic acids of this micro-aerobic responsiveness promotor and encoding transcription instrumentality is operatively connected, this transcriptional is just regulating the expression of one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route, and wherein the expression of this transcriptional raises under micro-aerobic fermentation condition.In some embodiments, this transcriptional is Gal4p, and one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route are operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10.

In some embodiments, this fermenting composition comprises the genetically modified host cell of a group in the substratum comprising carbon source, wherein this host cell comprises: (a) encodes one or more heterologous nucleic acids of one or more enzymes of mevalonic acid (MEV) approach, and it is operatively connected with Gal4p responsiveness promotor separately; (b) encode the nucleic acid of Gal4p, itself and micro-aerobic responsiveness promotor are operatively connected.In some embodiments, this host cell comprises the functional destruction of Gal80p further.In some embodiments, this micro-aerobic responsiveness promotor is saltant type DAN1 promotor.In some embodiments, this saltant type DAN1 promotor comprises the sequence being selected from lower group: SEQ ID NO:1,2,3,4,5,6,7,8,9 and 10.In some embodiments, this saltant type DAN1 promoter sequence comprises SEQ IDNO:1.In some embodiments, this saltant type DAN1 promoter sequence comprises SEQ ID NO:2.In some embodiments, this substratum comprises the dissolved oxygen concentration of 100%.In some embodiments, this substratum comprises and is less than about 20%, is less than about 15%, is less than about 10%, or is less than the dissolved oxygen concentration of about 5%.In some embodiments, this substratum comprises the dissolved oxygen concentration of about 0%.

In some embodiments, this fermenting composition comprises the genetically modified host cell of a group in the substratum comprising carbon source, wherein this host cell comprises coding for generating one or more heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of this allos, and wherein the expression of these one or more enzymes is just regulated by the activity of maltose responsiveness promotor.In some embodiments, one or more heterologous nucleic acids of one or more enzymes of this maltose responsiveness promotor and this coding enzymatic route are operatively connected, and wherein the expression of one or more enzymes of this enzymatic route reduces under maltose exists.In some embodiments, the heterologous nucleic acids of this maltose responsiveness promotor and encoding transcription instrumentality is operatively connected, the expression of one or more heterologous nucleic acids of one or more enzymes of this this coding enzymatic route of transcriptional negative regulator, wherein the expression of this transcriptional raises under maltose exists.In some embodiments, this transcriptional is Gal80p, this host cell comprises Gal4p further, and one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route are operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10.

In some embodiments, this fermenting composition comprises the genetically modified host cell of a group in the substratum comprising carbon source, wherein this host cell comprises: (a) encodes one or more heterologous nucleic acids of one or more enzymes of mevalonic acid (MEV) approach, and it is operatively connected with Gal4p responsiveness promotor separately; The nucleic acid of (b) coding Gal4p; (c) encode the nucleic acid of Gal80p, itself and maltose responsiveness promotor are operatively connected.In some embodiments, this maltose responsiveness promotor comprises the sequence being selected from lower group: pMAL1 (SEQ ID NO:12), pMAL2 (SEQ ID NO:13), pMAL11 (SEQ ID NO:14), pMAL12 (SEQ ID NO:15), pMAL31 (SEQ ID NO:16) and pMAL32 (SEQ ID NO:17).In some embodiments, this maltose responsiveness promoter sequence comprises pMAL32 (SEQ ID NO:17).In some embodiments, this substratum comprises at least 0.1% (w/v) maltose.In some embodiments, this substratum comprises 0.25% to 3% (w/v) maltose.In some embodiments, this substratum comprises and is no more than 0.08% maltose.

In some embodiments, this host cell is selected from lower group: fungal cell, bacterial cell, vegetable cell, and zooblast.In some embodiments, this host cell is yeast cell.In some embodiments, this non-alienation compound is selected from lower group: amino acid, lipid acid, isoprenoid, and polyketone.

In some embodiments, this host cell can generate isoprenoid and comprise the heterologous nucleic acids that at least one coding is selected from the Isoprenoid pathway enzyme of lower group: (a) condensation two molecule acetyl-coenzyme A is to form the enzyme of acetoacetyl-CoA; B () condensation acetoacetyl-CoA and another molecule acetyl-CoA are to form the enzyme of 3-hydroxy-3-methyl glutaryl base-CoA (HMG-CoA); C HMG-CoA is transformed into the enzyme of mevalonic acid by (); D mevalonic acid is transformed into the enzyme of mevalonic acid 5-phosphoric acid by (); E mevalonic acid 5-phosphoric acid is transformed into the enzyme of mevalonic acid 5-tetra-sodium by (); F mevalonic acid 5-tetra-sodium is transformed into the enzyme of IPP by (); G IPP is transformed into the enzyme of DMAPP by (); H () energy condensation IPP and/or DMAPP molecule are to form the polyisoprene synthase containing the polyisoprene compound more than five carbon; I () condensation IPP and DMAPP is to form the enzyme of GPP; The enzyme of (j) condensation two molecule I PP and a part DMAPP; K () condensation IPP and GPP is to form the enzyme of FPP; L () condensation IPP and DMAPP is to form the enzyme of GGPP; (m) condensation IPP and FPP is to form the enzyme of GGPP.

In some embodiments, this host cell comprises the heterologous nucleic acids that coding is selected from the enzyme of the modification polyisoprene of lower group further: lemonol (geraniol) synthase, phantol (linalool) synthase, limonene (limonene) synthase, myrcene (myrcene) synthase, ocimene (ocimene) synthase, α-pinene (α-pinene) synthase, beta-pinene (β-pinene) synthase, sabinene (sabinene) synthase, γ-terpinene (γ-terpinene) synthase, terpinolene (terpinolene) synthase, false indigo diene (amorphadiene) synthase, α-farnesene (α-farnesene) synthase, β-farnesene (β-farnesene) synthase, farnesol (farnesol) synthase, nerolidol (nerolidol) synthase, Patchoulicalcohol (patchouliol) synthase, nootkatone (nootkatone) synthase, rosin diene (abietadiene) synthase.

In some embodiments, this host cell comprises the multiple heterologous nucleic acids of all enzymes of coding mevalonate pathway.In some embodiments, this isoprenoid is selected from lower group: hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, and polyterpene.In some embodiments, this isoprenoid is C5-C20 isoprenoid.In some embodiments, this isoprenoid is sesquiterpene.In some embodiments, this isoprenoid is selected from lower group: rosin diene, false indigo diene, carene (carene), α-farnesene, β-farnesene, farnesol, lemonol, yak base lemonol (geranylgeraniol), isoprene (isoprene), phantol, limonene, myrcene, nerolidol, ocimene, Patchoulicalcohol, beta-pinene, sabinene, γ-terpinene, terpinolene and valencene (valencene).

In some embodiments, this host cell can generate polyketone and comprise the heterologous nucleic acids of at least one coding polyketide synthases, and wherein this polyketide synthases is selected from lower group: the enzyme of at least one item and acyl carrier protein in (a) condensation acetyl-CoA and malonyl--CoA; B () condensation is selected from first reactant of acetyl-CoA and malonyl--CoA and is selected from second reactant of malonyl--CoA or methylmalonyl-CoA to form the enzyme of polyketone product; C beta-keto chemical group on polyketide is reduced into the enzyme of beta-hydroxy group by (); D () makes the alkane chemical group in polyketide dewater with the enzyme generating alpha-beta-unsaturated olefin; E alpha-beta-reduction in polyketide is become the enzyme of saturated alkane by (); (f) from the enzyme of acyl carrier protein hydrolyzed polyketone compound.

In some embodiments, this polyketone is for having antibiosis, antimycotic, and the lipid of at least one in anti-tumor activity.In some embodiments, this polyketone is selected from lower group: macrolide, and biocide is antimycotic, T suppression cell compound, Anticholesterolemic compound, anti-parasitic compound, suppresses coccidia compound, animal growth promoter and sterilant.

In some embodiments, this host cell can generate lipid acid and comprise the heterologous nucleic acids of at least one encode fatty acid synthetic enzyme, and wherein this fatty acid synthetase is selected from lower group: at least one item in acetyl-CoA and malonyl--CoA is covalently attached to the enzyme of acyl carrier protein (ACP) by (a); B () condensation acetyl-ACP and malonyl--ACP is to form the enzyme of acetoacetyl-ACP; C double bond that () is reduced in acetoacetyl-ACP with NADPH is to form the oh group in D-3-maloyl group hydroxylase-ACP; D enzyme that () makes D-3-maloyl group hydroxylase-ACP dewater to produce the double bond between β-and γ-carbon forming crotonyl-ACP; E () reduces crotonyl-ACP to form the enzyme of butyryl radicals-ACP with NADPH; (f) from acyl carrier protein hydrolysis C ₁₆acyl compounds is to form the enzyme of palmitinic acid.In some embodiments, this lipid acid is selected from lower group: palmitinic acid (palmitate), palmityl CoA (palmitoyl CoA), Zoomeric acid (palmitoleic acid), fragrant juniperic acid (sapienic acid), oleic acid (oleic acid), linolic acid (linoleic acid), alpha-linolenic acid (α-linolenic acid), arachidonic acid (arachidonic acid), timnodonic acid (eicosapentaenoic acid), erucic acid (erucic acid), with docosahexenoic acid (docosahexaenoic acid).

Accompanying drawing is sketched

Fig. 1 is provided for the schematic diagram of mevalonic acid (" the MEV ") approach generating isopentenyl diphosphate (" IPP ").

Fig. 2 provides IPP and dimethylallylpyrophosphate (" DMAPP ") to be transformed into yak base tetra-sodium (" GPP "), farnesyl pyrophosphate (" FPP "), and yak base yak base tetra-sodium (" GGPP ") schematic diagram.

Fig. 3 display can generate the strain degeneration (namely non-alienation compound generates and declines in time) of a group yeast host cell of non-alienation compound farnesene.

Fig. 4 is provided for the schematic diagram controlling a kind of exemplary hypoxemia switch based on GAL regulon that allos non-alienation compound generates in host cell.

Fig. 5 is provided for the schematic diagram controlling a kind of exemplary maltose switch based on GAL regulon that allos non-alienation compound generates in host cell.

The result that Fig. 6 provides proves can generate isoprenoid farnesene and be included in micro-aerobic responsiveness promotor (" low O ₂switch ") just regulating under the host cell of MEV approach at high O ₂in condition, (rocker) generates the farnesene of very low amounts, and at low O ₂in condition (low RPM shaking flask), generate substantive be increased to MEV approach constructive expression can not the generation of the switch parent strain level of mating.

Compared with result that Fig. 7 provides proves to produce the generation of bacterial strain with the composition running through the construction phase and generate farnesene, when implementing construction phase (the realizing "Off" state thus) of fermenting under aerobic condition, isoprenoid farnesene can be generated and be included in low O ₂the host cell of the MEV approach under switch is just regulating shows that in long hair ferment runs the farnesene of improvement generates stability.

Result that Fig. 8 provides proves to generate isoprenoid farnesene and the host cell (1.3%) under maltose exists of MEV approach under being included in maltose responsiveness promotor (" maltose switch ") negative regulator generates the farnesene of very low amounts, and under maltose disappearance, generate substantive be increased to close to MEV approach constructive expression can not the level of generation of switch parent strain.

Compared with the parent strain that result proof and composition that Fig. 9 provides generate farnesene, isoprenoid farnesene can be generated and be included in micro-aerobic responsiveness promotor (" low O ₂switch ") just to regulate down or the host cell of MEV approach under maltose responsiveness promotor (" maltose switch ") negative regulator has the growth velocity of improvement during compound generates "Off" state.

Compared with result that Figure 10 provides proves to produce the generation of bacterial strain with the composition running through the construction phase and generate farnesene, when implementing construction phase (the realizing "Off" state thus) of fermenting under existing at maltose, isoprenoid farnesene can be generated and the host cell of MEV approach under being included in maltose switch negative regulator shows that in long hair ferment runs the farnesene of improvement generates stability.

The result that Figure 11 provides is that maltose susceptibility promotor pMAL11 proves that (a) is to the maltose of the difference amount in substratum and the susceptibility to mixing feed, and (b) after the preventing of maltose, becomes the switch of "On" state in "Off" state under maltose disappearance.

The result that Figure 12 provides is that maltose susceptibility promotor pMAL12 proves that (a) is to the maltose of the difference amount in substratum and the susceptibility mixing feed, and (b) after the preventing of maltose, becomes the switch of "On" state in "Off" state under maltose disappearance.

The result that Figure 13 provides is that maltose susceptibility promotor pMAL31 proves that (a) is to the maltose of the difference amount in substratum and the susceptibility mixing feed, and (b) after the preventing of maltose, becomes the switch of "On" state in "Off" state under maltose disappearance.

The result that Figure 14 provides is that maltose susceptibility promotor pMAL32 proves that (a) is to the maltose of the difference amount in substratum and the susceptibility mixing feed, and (b) after the preventing of maltose, becomes the switch of "On" state in "Off" state under maltose disappearance.

Detailed Description Of The Invention

1. define

As used in this article, refer to can the material of natural generation in host cell or process for term " endogenous ".

As used in this article, phrase " functional destruction " such as target gene means change target gene thus reduces the activity of the protein of being encoded by this target gene in host cell.Similarly, " functional destruction " such as target protein means change target protein thus reduces the activity of this albumen in host cell.In some embodiments, the activity of the target protein of being encoded by target gene in host cell is eliminated.In other embodiments, the activity of the target protein of being encoded by target gene in host cell is reduced.Functional destruction target gene, by deleting gene all or in part, makes genetic expression eliminate or reduces or the activity of gene product eliminated or reduces to realize.Functional destruction target gene is also by the regulatory element of mutator gene, and the promotor of such as gene, makes to express and eliminate or reduce, or by the encoding sequence of mutator gene, the activity of gene product is eliminated or reduces to realize.In some embodiments, functional destruction target gene causes the whole open reading-frame (ORF) of target gene to be removed.

As used in this article, term " genetically modified " represents that host cell comprises heterologous nucleotide sequence.

As used in this article, term " allos " refers to not find at nature under normal circumstances.Term " heterologous compounds " refers to that generating the cell of compound does not generate this compound under normal circumstances, or the level generating compound is not its level of this Hemapoiesis under normal circumstances.

As used in this article, phrase " isodynamic enzyme " refers to the enzyme that do not find in the given cell of occurring in nature under normal circumstances.Following enzyme contained in this term:

(a) to given cell be external source (namely by and non-natural be present in host cell or and non-natural to be present in host cell in given background nucleotide sequence coded); With

(b) in host cell natural (such as this enzyme be by be endogenous nucleotide sequence coded to cell) of finding but generate (being such as greater than or less than the natural amount found) with non-natural amount in host cell.

As used in this article, phrase " is operatively connected " the functional connection referred between nucleotide sequence, makes the expression of the functional control coding sequence of promotor and/or regulatory region connected.

As used in this article, term " output " refers generally to the amount of the non-alienation compound generated by the genetically modified host cell provided herein.In some embodiments, output states the non-alienation compound productive rate of chief cell all night.In other embodiments, output states the productivity that chief cell all night generates non-alienation compound.

As used in this article, term " productivity " refers to the non-alienation compound production of host cell, and the fermention medium (by volume) stating every deal of cultivating host cell as is (per hour) amount (by weight) of non-alienation compound of generating in time.

As used in this article, term " promotor " refers to give, and activates or strengthen synthesis or the natural derivative nucleic acid that DNA encoding sequence is expressed.Promotor can comprise one or more specific transcriptionals and regulate sequence to strengthen the expression of encoding sequence further and/or to change space expression and/or temporal expressions.Promotor can be positioned at the 5'(upstream of encoding sequence at the control).Distance between promotor and the encoding sequence that will express can be substantially equal to the distance between the native sequence nucleic acid of promotor and its control.Know as this area, this distance can hold change, is not activated subfunction and loses.This modulated promotor used herein is generally being in admissibility environment (such as micro-aerobic fermentation condition, or under maltose exists) in time allow encoding transcription instrumentality (such as activator, such as pGal4, or repressor, such as pGal80) nucleotide sequence transcribe, but the nucleotide sequence stopping encoding transcription instrumentality when being in inadmissibility environment (such as aerobic fermentation condition, or under maltose disappearance) is transcribed.

Phrase " strain stability " refers generally to the stability that genetically modified host cell described herein generates heterologous compounds in the cultivation period extended.Especially, stability refers to the inner ability maintaining the favourable generating feature (i.e. high yield (g of compound every gram substrate) and/or productivity (gram often liter of fermention medium is per hour)) of non-alienation tunning of incubation time (such as 3 to 20 days) that microorganism is extending.Genetic stability (namely producing the appointment gene frequency generating relevant gene in micropopulation with product changes little of unconverted tendency in time) plays Main Function in lasting output of products.

Term " productive rate " refers to the non-alienation compound production of host cell, the amount of the non-alienation compound that the carbon source stating every deal that host cell consumes as generates, by weight.

2. the switch that oxygen sensitivity promotor generates as heterologous compounds is combinationally used with micro-aerobic fermentation

In some embodiments, the method and composition provided herein utilizes oxygen sensitivity promotor under micro-aerobic fermentation condition, drive the isodynamic enzyme that can realize the generation of non-alienation compound to express in genetically modified host cell.When carrying out the fermentation of host cell under aerobic fermentation condition, non-alienation compound generates substantive reduction or closedown; When fermentation condition be micro-aerobic time, non-alienation compound generates to be opened or raises.So, genetically modified cell described herein can use the switch that hypoxia condition generates as non-alienation compound.Especially, the opportunity that non-alienation compound generates is controlled to only carbon stream is altered course into cell maintenance and biomass during wanting to occur in non-productive stage when generating.This more efficient carbon uses the metabolism burden greatly reducing host cell, improves the stability of heterologous gene, reduces strain degeneration, and contribute to better cell population health and viability.Thus, the method provided herein and genetically modified host cell utilize hypoxemia fermentation condition as switch to realize "Off" and the "ON" stage of the improvement zymotechnique of the non-alienation compound generation of allos.

In a first step (i.e. " construction " stage, step (a)), under aerobic condition, namely wherein provide oxygen with non-limiting amounts, genetically modified host cell is grown in growth or " construction " substratum.In second step (i.e. " generation " stage, step (b)), ferment under micro-aerobic condition, this serves as non-genetic switch and carrys out the non-alienation compound generation of substantive strengthening.Initial growth under complete aerobic condition guarantees the energy requirement meeting cell, increases the biomass of cell fast simultaneously.After this, convert micro-aerobic condition to and can synthesize non-catabolite.

2.1 oxygen sensitivity DAN1 promotors

In some embodiments, for regulating in the method provided in this article, can to realize the useful oxygen sensitivity promotor of expression of enzymes that non-alienation compound generates be DAN1 promotor and homologue thereof and variant (SEQ ID NO:1-11).In some embodiments, DAN1 promotor is from yeast saccharomyces cerevisiae.Wild-type DAN1 promotor (SEQ ID NO:1) non-activity but under anaerobic highly have activity under aerobic condition.See such as Kwast et al., J Bacteriol.184 (1): 250-265 (2002); Piper et al., J Biol Chem 277 (40): 37001-37008 (2002); And ter Linde et al., J Bacteriol.181 (24): 7409-7413 (1999).

(see Lai et al., Mol Cell Biol.25 (10): 4075-4091 (2005) in the large group gene that yeast saccharomyces cerevisiae DAN/TIR gene raises during adapting to aerobic growth; Sertil et al., Gene192 (2): 199-205 (1997); And Tai et al., J Biol Chem.280 (1): 437-447 (2005)).The cell walls Mannoproteins that these genes encodings play a significant role in cell walls permeability.The dynamics range of these genetic expressions is that anoxybiosis starts latter 30 minutes to 3 hours (see Abramova et al., JBacteriol.183 (9): 2881-2887 (2001)).It seems and adapt to complicated apoptosis cell wall transformation occurs during anoxybiosis, as shown in the fact that the main aerobic cell walls Mannoproteins of being encoded by CWP1 and CWP2 is under those circumstances replaced by the aerobic counterpart of DAN/TIR genes encoding by them.

By blasting nitrogen to culture to cut down oxygen, achieve the harsh anoxybiosis (see Cohen et al., Nucleic Acids Res 29 (3): 799-808 (2001)) that effectively induction wild-type DAN1 promotor requires.But the people such as Nevoigt develop and are relating to derivable a series of DAN1 promoter mutation body (SEQ ID NO:1-10) under the simple condition eliminated or reduce ventilation.See Nevoigt et al., Biotechnology and Bioengineering 96 (3): 550-558 (2007); And U.S. Patent application No.2007/0178505, completely include its content by addressing.

So, in some embodiments, DAN1 promotor useful in the method provided in this article is those that record in U.S. Patent application No.2007/0178505, and comprises and comprise SEQ ID No:1,2,3,4,5,6,7,8,9, or the promotor of 10.In some embodiments, DAN1 promotor useful in the method provided in this article comprises SEQ ID No:1, and 2,3,4,5, or 6.In some embodiments, DAN1 promotor useful in the method provided in this article comprises SEQ IDNo:1 or 2.In some embodiments, DAN1 promotor useful in the method provided in this article comprises SEQ ID No:1.In some embodiments, DAN1 promotor useful in the method provided in this article comprises SEQ ID No:2.

In another embodiment, sudden change is comprised in one or more in the following position of SEQ ID No:11 of DAN1 promotor useful in the method provided in this article: 1-56; 66-139; 148-232; 245-283; 290-293; 301-302; 310; 322-326; 334-347; 357-371; 380-450; Or 458-551.According to this aspect and in one embodiment, sudden change is positioned at position: 4, 7, 15, 18, 19, 21, 22, 26, 28, 36, 40, 53, 56, 60, 63, 66, 74, 75, 78, 86, 99, 122, 132, 135, 136, 149, 153, 162, 164, 165, 171, 172, 176, 187, 196, 198, 201, 205, 207, 211, 216, 226, 228, 233, 234, 237, 241, 260, 269, 274, 277, 280, 281, 285, 296, 299, 303, 307, 308, 310, 313, 322, 327, 331, 332, 337, 338, 343, 344, 346, 366, 368, 373, 375, 376, 381, 384, 386, 390, 391, 392, 396, 397, 402, 404, 422, 427, 428, 429, 432, 434, 439, 445, 467, 469, 470, 477, 480, 490, 492, 508, 511, 514, 518, 528, or its combination.In one embodiment, the sudden change being positioned at these positions becomes any Nucleotide except wild type nucleotide, and in another embodiment, the sudden change being positioned at each position becomes described specific nucleotide hereinbelow.

In another embodiment, DAN1 promotor useful in the method provided in this article comprises following sequence, and this sequence comprises following replacement: sequence (a) nucleotide position 4,15 shown in SEQ ID NO:11,19,36,53,56,60,66,74,75,78,86,99,132,136,176,201,205,207,216,226,228,269,277,281,285,299,303,310,327,331,332,375,376,390,428,434,467,477,480,508,511, or the T C of its combination replaces; (b) nucleotide position 7,18,26,40,122,135,149,153,162,164,165,171,172,187,196,211,233,234,237,241,260,274,280,308,313,322,337,343,344,346,366,368,381,384,386,396,397,402,404,422,427,429,432,445,470,490,492, or the A G of its combination replaces; C the C A of () nucleotide position 21 replaces; (d) nucleotide position 237,338, the A C of 469,514,518 replaces; (e) nucleotide position 28,296,307,373,392,528, or the C T of its combination replaces; (f) nucleotide position 22,63,391,439 or its combination G A replace; G the T G of () nucleotide position 198 replaces; Or its any combination.

In another embodiment, DAN1 promotor useful in the method provided in this article with promotor part in structure or functionally homology of the DAN1 promotor part of sudden change described herein in comprise sudden change.In another embodiment, promotor useful in the method provided in this article with the promotor of DAN1 promotor homology in comprise sudden change.In one embodiment, homologous promoter or its part are derived from yeast saccharomyces cerevisiae/Saccharomyces cerevisiae (S.cerevisiae) sequence, and in another embodiment, they are derived from other (sugar) yeast belong (Saccharomyces) species, and in another embodiment, they are derived from (sugar) Saccharomycetaceae (Saccharomycetaceae), and in another embodiment, they are derived from (sugar) Saccharomycetes (Saccharomycetales), and in another embodiment, they are derived from (sugar) yeast guiding principle (Saccharomycetes), and in another embodiment, they are derived from (sugar) yeast subphylum (Saccharomycotina), and in another embodiment, they are derived from Ascomycota (Ascomycota), and in another embodiment, they are derived from fungal species.In another embodiment, the oxygen dependence similar to DAN1 promotor is shown to the promotor of DAN1 promotor homology.Those skilled in the art can use this area Routine methods to determine the oxygen dependence of promotor.Those skilled in the art's routine uses instrument known in the art such as sequence alignment to carry out the determination of homologous promoter or promoter region.

In one embodiment, the homologous promoter of DAN1 is DAN2, DAN3, DAN4, TIR1, TIR2, TIR3, or TIR4.In another embodiment, the homologous promoter of DAN1 is CYC1, CYC7, ANB1, COX5b, ERG11, MOX1, MOX2, MOX4/UPC2, ROX7/MOT3, or ROX1 promotor.

In one embodiment, sudden change can in the promotor part corresponding with aerobic response element binding site, and it is AR1 or AR2 in one embodiment, and in another embodiment, sudden change can in Mot3 or Rox1 binding site.

2.1.1 the target of DAN1 promotor adjustment

In some embodiments, the method provided herein utilizes genetically modified host cell, and it comprises coding for generating the heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of allos.In some embodiments, the expression of one or more enzymes is under saltant type DAN1 promotor directly controls.In other words, one or more heterologous nucleic acid sequence of one or more enzymes of coding enzymatic route are operatively connected (being namely positioned at 3 ') with saltant type DAN1 promotor separately, and saltant type DAN1 promotor drives one or more heterologous nucleic acids described under micro-aerobic condition, and each is expressed.

In other embodiments, the expression of one or more enzymes of enzymatic route is by saltant type DAN1 promotor indirect regulation.Such as, the indirect regulation of one or more enzymes of approach can realize as follows, be operatively connected by saltant type DAN1 promotor and a kind of heterologous transcription instrumentality, the expression of the latter directly regulates the expression of one or more enzymes (such as all members) of approach then.

GAL regulon in yeast provides and can combine with saltant type DAN1 promotor described herein activation utilized, and prevents the exemplary regulating networks of one of son and promotor.Via expression GAL gene input semi-lactosi and cell interior metabolism it, yeast can utilize semi-lactosi as carbon source.GAL gene comprises structure gene GAL1, GAL2, GAL7, (they are encoding galactose kinases respectively with GAL10 gene, galactose permease, α-D-galactose-1-phosphate uridyl transferring enzyme, and uridine diphosphate galactose-4-epimerase) and regulatory gene GAL4, GAL80, and GAL3.For GAL1, GAL2, GAL7, and the expression of GAL10 gene, GAL4 gene product is positive regulator (namely activating son) and GAL80 gene product is down regulator (namely preventing son).Gal4p is by carrying out activated transcription in conjunction with upstream activating sequence (UAS) (those of such as GAL structure gene, namely in pGAL1, pGAL7 and pGAL10 promotor).Under semi-lactosi disappearance, interact due to Gal80p and Gal4p and stop Gal4p transcriptional activity, usually detecting that little structural protein (Gal1p, Gal2p, Gal7p, and Gal10p) are expressed.But under semi-lactosi exists, Gal3p and Gal80p interacts, and removes Gal80p preventing Gal4p.This allows the gene (such as GAL1, GAL2, GAL7, and GAL10 gene product) in Gal4p binding sequence downstream to express.

So, in some embodiments, one or more GAL4 are activated promotor (such as pGAL1, pGAL7, and/or pGAL10) to be operatively connected with one or more enzymes for the enzymatic route that generates the non-alienation compound of allos and for driving it to express, and by the expression of saltant type DAN1 promoters driven GAL4 gene product described herein.Thus, under micro-aerobic condition by the expression of one or more enzymes of Gal4p ways of regeneration.In some these type of embodiments, use the known technology being used for gene disruption reduce or eliminate the expression of GAL80 gene, make no longer there is Gal80p and carry out negative regulator Gal4p activity, no matter the oxygen condition of fermentation.In some embodiments, the heterologous nucleic acids of the involved saltant type DAN1 promotor of natural pGAL4 promotor is replaced.In some embodiments, host cell comprises following heterologous nucleic acids, and it comprises the nucleic acid of coding Gal4p, is operatively connected with the heterologous nucleic acids comprising saltant type pDAN1 promotor.In one embodiment, the encoding sequence of saltant type DAN1 promotor and Gal4p is operatively connected, and is operatively connected for the encoding sequence of one or more enzymes (such as all members) of the enzymatic route that generates allos non-alienation compound and GAL4 responsiveness promotor.In some embodiments, GAL4 responsiveness promotor is pGAL1.In some embodiments, GAL4 responsiveness promotor is pGAL7.In some embodiments, GAL4 responsiveness promotor is pGAL10.

Aerobic and micro-good oxygen amount of 2.2 oxygen

In some embodiment of the method provided in this article, cultivate under not by the condition of oxygen restriction and aerobic condition during the construction phase or maintain cell, continuing cultivate under oxygen restrictive condition and micro-aerobic or anaerobic condition or maintain cell.

It can be challenging for maintaining complete aerobic condition, and particularly in Large scale processes, reason is the restriction of mass transfer and the relatively low in aqueous solubleness of oxygen.Such as, if use air to blast tank, it is so 9 milligrams per liter in the solubleness of 20 DEG C of oxygen in water.If use pure oxygen to replace air, so solubleness is increased to 43 milligrams per liter.In arbitrary situation, (blast air or pure oxygen), the oxygen of this tittle is exhausted in a few second by activated and concentrated micropopulation, unless oxygen without interruption.Comparatively speaking, other nutraceutical amount of period identical period (a few second is such as less than one minute) cell use can be ignored compared with huge concentration.We have found the host cell generating allos non-alienation compound can tolerate the oxygen restriction of some periods and still generate high-caliber isoprenoid compounds.By designing at tank, oxygen demand reduces, and the lower aspect like this of cost of energy of ventilation is saved, and this handiness allows more cost effective technique.

When the specific growth rate of host cell is less than maximum specific growth rate there is oxygen restriction in (now oxygen is not restrictive (such as excessive provide)).Specific growth rate is the cell growth rate of per unit biomass time per unit, and has the unit (1/t) of time inverse.Cell maximum specific growth rate in the medium relates to the impact of concentration of substrate on growth velocity, is oxygen in this case.Usually, cell can slowly grow at low-level substrate, and along with the substrate-level rising in substratum, cell growth rate also raises.But cell growth rate does not continue unlimited rising, and at high-caliber substrate, the given rising of amount of substrate can cause the more and more less rising of cell growth rate.Therefore, growth velocity finally reaches restriction, and this is often referred to as maximum specific growth rate.

The theoretical treatment of the dependency between the growth velocity in culture well known to a person skilled in the art, and be called Monod equation.See such as Pirt, Principles of Microbe and CellCultivation, Wiley, NY, 1975, pages 4-10.In this theoretical treatment, high specific speed is until reach the substrate of infinite horizontal, the progressive restriction never reached.But in practice, maximum specific growth rate can consider what condition (such as substrate-level, such as oxygen) in investigation was supported to obtain during the fastest initial growth speed.Such as, in fedbatch reactor, treat using all substrates (such as nutrition and oxygen) of excessive supply growth needs with in the starting condition that the temperature of host cell the best is occurred to for fermentation as the condition of maximum growth rate.See such as Lee et al. (1996) TrendsBiotechnol.14:98-105 and Korz et al. (1995) J Biotechnology 39:59-65.Maximum specific growth rate is sometimes also referred to as unrestricted growth.

In one approach, the oxygen concn in oxygen restricted passage substratum quantizes, and states in dissolved oxygen concentration (DOC).DOC in substratum can be less than about 20%, is less than about 15%, is less than about 10%, and is less than about 5%.In other embodiments, DOC is about 0% or lower than detection level.But, because the relatively very fast oxygen consumed of cell, so zero DOC can mean that under anaerobic (anaerobic) culturing cell or cell consumption oxygen soon must as its supplies.In another approach, at oxygen uptake rate (OUR; The speed of often liter of substratum cell consumption oxygen) aspect statement cell oxygen use to distinguish this two kinds of possibilities.Suitable oxygen uptake rate comprises and is less than about 50 mmoles, is less than about 40 mmoles, is less than about 30 mmoles, is less than about 20 mmoles and often rises substratum, or is less than about 10 mmoles and often rises substratum.Or, can use than oxygen uptake rate (SOUR, namely OUR is divided by cell density) when preferred value standardized about cell density.The amount of often liter of fermented liquid microbe or the density of microbe can be measured by the weight measuring the microbe be separated from the fermention medium of given volume.A kind of conventional measurement is often liter of fermention medium dry cell weight.The another kind of method that can be used in monitoring fermentation in fermentation is carried out measures the optical density(OD) of substratum.Conventional method measures the optical density(OD) at wavelength 600nm place, is called OD ₆₀₀, or OD.OD can be relevant with the density of the organism of particular type in defined medium, but the specific dependency between the microorganism scale of construction of OD and every volume generally can not be suitable in all types of substratum between all types of organism.By measuring OD and dry cell weight in a cell density scope, working curve can be produced.In some cases, can these be used to calibrate in the different fermentations of same or similar microbe in same or similar substratum.Suitable ratio oxygen uptake rate comprises and is less than about 30 mmoles, is less than about 25 mmoles, is less than about 20 mmoles, is less than about 15 mmoles, is less than about 10 mmoles, or it is per hour to be less than about 5 mmoles every gram of dry cell weight.

Dissolved oxygen content can be had to maintain Growth of Cells and to maintain cellular metabolism, for generating non-alienation compound as required during culturing process according to construction phase or production phase maintain base.Currently known methods can be used to monitor the oxygen concn of substratum, such as via use oxygen electrode.Means known in the art can be used to add oxygen to substratum, stir and ventilation via to substratum, by stirring, shake or air-blowing.Use air to describe substratum ventilation although related to herein, other oxygen source can be used.Useful especially is use the oxygen volume fraction charge air conditioning containing larger than the oxygen volume fraction in ambient air.In addition, this type of charge air conditioning can comprise other gas culture not being had to negative impact.

In some embodiments, micro-aerobic condition is realized by blasting nitrogen (such as high-purity nitrogen (99.8%)) to culture.In some embodiments, micro-aerobic condition is realized by culturing cell in airtight vessel (such as screw cap vial and flask, like this).Because residual dissolved oxygen is consumed during Growth of Cells, so these conditions sharply reduce but do not exhaust oxygen operability completely during cell growth process.In some embodiments, can rely on sufficient quantity mixing air and vector gas to realize micro-aerobic condition.Or, suitable low flow velocity gas can be blasted.Oxygen electrode or other appropriate device any can be used to monitor the oxygen level in fermention medium, and the flow velocity of adjustments of gas mixture is to guarantee that the oxygen horizontal dimension in fermentation fluid is held in constant level.Outside the change of inlet gas flow velocity or inlet gas composition, the speed oxygen supply of large-scale culture (so reduce) can also be stirred or by adding more feeds to improve cell density (and therefore higher oxygen demand) or its combination generates micro-aerobic condition by reducing.

In some embodiments, dissolved oxygen (dO ₂) can by running through major part fermentation to host cell feed sugar to keep dO ₂concentration is in and can not controls by detection level.In some embodiments, can via advertising pressurized gas and mechanical agitation fermention medium to supply oxygen.In some embodiments, be about 50 to 200mmol O with scope ₂/ L/h, such as about 50,60,70,80,90,100,110,120,130,140,150,160,170,180,190 or 200mmol O ₂the speed supply oxygen of/L/h.In one particular embodiment, with about 110mmol O ₂the speed supply oxygen of/L/h.In order to utilize all these available oxygen, feed sugar must be enough to guarantee via its metabolism by all available dO in substratum soon ₂be transformed into H ₂o, generates enough NADH.In order to ensure this point, with than O ₂stoichiometry need slightly faster speed feed sugar, make to generate some ethanol.Termly, feed sugar does not consume the ethanol of generation again with Induced cultures, and this process also needs culture to consume O ₂.Once ethanol exhausts, dO ₂concentration raises fast, sends the signal that culture exhausts oxidizable carbon.By this dO of probe measurement ₂peak (spike), and technique restarts feed sugar, makes dO ₂returning can not detection level.

Suppose that the environment mixed does not have spatial gradient and ignores negligible dilution item (since enter the O of tank ₂with the O entering gas phase ₂compare and can ignore), the one-level differential equation describing the change of dissolved oxygen in fermentor tank is as follows:

\frac{d ({dO}_{2})}{dt} = k_{l} a ({dO}_{2 sat} - {dO}_{2}) {- q}_{O 2} [X]

Wherein:

DO ₂for the dissolved oxygen concentration in reactor during time t

K _lfor O ₂vapor-liquid coefficient of mass transfer

A is that foam surface amasss the ratio with liquid volume

DO _2satfor the O in liquid phase ₂equilibrium concentration, with the temperature of technique, pressure, and gas phase O ₂concentration is corresponding

Q _o2for the ratio oxygen consumption rate (mmol/g dw/h) of gram biomass

[X] is the biomass concentration in reactor during time t

Can simplify this equation and affect dissolved oxygen concentration to have highlighted two important documents: oxygen transfer rate (OTR), it is the mass transfer feature (k of substratum _l), the motivating force (dO of gas transport surface-area (a) and mass transfer _2sat-dO ₂) function; With oxygen uptake rate (OUR), it is that biomass concentration ([X]) and biomass consume O ₂there is how soon (qO ₂) function.So now only:

\frac{d ({dO}_{2})}{dt} = OTR - OUR

These equations can be used in help and understand dO ₂time overview.

3. the purposes of switch that generates as heterologous compounds of maltose responsiveness promotor and maltose operative combination

In some embodiments, the method and composition provided herein utilizes maltose responsiveness promotor, with the maltose content operative combination in fermention medium, for directly or indirectly regulating the isodynamic enzyme expression that can realize non-alienation compound and generate in genetically modified host cell.

In one embodiment, when (such as at least 0.1% maltose) under existing at maltose carries out host cell fermentation, non-alienation compound generates substantive reduction or closedown, and at that time in fermention medium the amount of maltose reduce or eliminate and, non-alienation compound generates to be opened or raises.So, in some embodiments, genetically modified cell described herein comprises the heterologous organisms route of synthesis gene regulated by maltose responsiveness promotor, can use the switch that the maltose in fermention medium generates as non-alienation compound.Control the opportunity that non-alienation compound generates, only carbon stream is altered course into cell maintenance and biomass during wanting to occur in non-productive stage when generating.This more efficient carbon uses the metabolism burden greatly reducing host cell, improves Growth of Cells, improves the stability of heterologous gene, reduce strain degeneration, and contribute to better cell population health and viability.

In some embodiments, fermentation process comprises two-step process, and it utilizes maltose as switch to realize "Off" and "ON" stage.(i.e. " construction " stage in the first step generated not wanting compound, step (a)), genetically modified host cell grows in the growth comprising maltose with the amount being enough to the genetic expression of induction under maltose responsiveness promotor controls or " construction " substratum, and the gene product of induction plays the non-alienation compound generation of negative regulator.(i.e. " generation " stage in second step, step (b)), ferment in the substratum comprising carbon source, wherein maltose lacks or measures so low that to be enough to make maltose responsiveness promotor no longer include activity, and the non-alienation compound of the allos of host cell generates unlatching or raises.

In other embodiments, maltose responsiveness promotor can be operatively connected with one or more heterologous nucleic acids of one or more enzymes of coding enzymatic route, and the maltose that there is activation amount in substratum improves the expression of one or more enzymes of enzymatic route.By this way, the positive regulator that maltose responsiveness promotor can be routed to as non-alienation compound generation works.

3.1 maltose responsiveness promotors

In preferred embodiments, maltose responsiveness promotor useful in the method and composition provided in this article promotes the DNA encoding sequence transcribes be operatively connected under maltose exists.In some embodiments, exchanging in energy-conservation enough method and compositions provided in this article the maltose responsiveness promotor realizing expression of enzymes that non-alienation compound generates useful is any maltose responsiveness promotor known in the art.In some embodiments, maltose responsiveness promotor is selected from lower group: pMAL1 (SEQ IDNO:12), pMAL2 (SEQ ID NO:13), pMAL11 (SEQ ID NO:14), pMAL12 (SEQ ID NO:15), pMAL31 (SEQ ID NO:16) and pMAL32 (SEQ ID NO:17).In specific embodiments, maltose susceptibility promotor is pMAL32 (SEQ ID NO:17).

Other useful maltose responsiveness promotor useful in the method and composition provided in this article can derived from the regulating networks of the maltose fermentation system of yeast saccharomyces cerevisiae.Maltose fermentation in sugar yeast species requires at least one item in the MAL locus that existence five kinds is not chain: MAL1, MAL2, MAL3, MAL4, and MAL6.These locus are each freely involves the glycometabolic gene complex composition of Fructus Hordei Germinatus; This complex body comprises maltin, maltose permease, and the activator of these genes.At MAL6 locus place, activator is by MAL63 genes encoding.Mal63p is that the DNA of the maltose dependency derived need of the MAL structure gene of encoding mannose permease and maltin is in conjunction with transcriptional activator.

MAL activator intermediate complex body is stable under inductor maltose disappearance, but interpolation maltose causes can induce MAL activator to discharge from complex body with the activity form that can carry out DNA combination and transcriptional activation.See such as Ran, F.and Michels., C.A., J.Biol.Chem.285 (18): 13850-13862 (2010).The binding site having characterized MAL63 albumen in the MAL61-62 promotor that difference transcribes is the upstream activating sequence of MAL gene.See such as Ni, B.andNeedleman, R., " Identification of the Upstream Activating Sequence of MAL andthe Binding Sites for the MAL63 Activator of Saccharomyces cerevisiae; " Molecular and Cellular Biology 10 (7): 3797-3800 (1990), completely includes its content by addressing.

Other useful maltose responsiveness promotor useful in the method and composition provided in this article can derived from the regulating networks of colibacillary maltose/maltodextrin metabolic system.MalT nucleic acid encoding MalT, it is four kinds of maltose responsiveness promotor (P _pQ, P _eFG, P _kBM, and P _s) positive regulator.The combination of malT and mal promotor produces the expression system closely regulated, and it has demonstrated as the strong promoter work by adding maltose induction.See such as Schleif, " Two PositivelyRegulated Systems; ara and mal, " pp.1300-1309 in Escherichia coli andSalmonella Cellular and Molecular Biology, Second Edition, Neidhardt et al., eds., ASM Press, Washington, D.C., 1996; And Boos, W.and Shuman, H., " Maltose/Maltodextrin System of Escherichia coli:Transport; Metabolism andRegulation; " Microbiology and Molecular Biology Reviews, 62 (1): 204-229 (1998), completely include its content accordingly by addressing.

Other useful maltose responsiveness promotor useful in the method and composition provided in this article comprises Berkner et al., " Inducible and constitutive promoters for genetic systems inSulfolobus acidocaldarious, " Extremophiles 14:249-259 (2010); And U.S. Patent No. 5,824, in 545 record those.

3.1.1 the target of maltose responsiveness promotor adjustment

In some embodiments, the method provided herein utilizes genetically modified host cell, and it comprises coding for generating the heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of allos.In some embodiments, the expression of one or more enzymes is under maltose responsiveness promotor described herein directly controls.In other words, one or more heterologous nucleic acid sequence of one or more enzymes of coding enzymatic route are operatively connected (being namely positioned at 3 ') with maltose responsiveness promotor separately, and maltose responsiveness promotor drives one or more heterologous nucleic acids described under maltose exists, and each is expressed.

In other embodiments, the expression of one or more enzymes of enzymatic route is by maltose responsiveness promotor indirect regulation.Such as, the indirect regulation of one or more enzymes of approach can realize as follows, be operatively connected by maltose responsiveness promotor and a kind of heterologous transcription instrumentality, the expression of the latter directly regulates the expression of one or more enzymes (such as all members) of approach then.As detailed above, the GAL regulon in yeast provides and can combine with maltose responsiveness promotor described herein activation utilized, and prevents the exemplary regulating networks of one of son and promotor.

In some embodiments, one or more GAL4 are activated promotor (such as pGAL1, pGAL7, and/or pGAL10) to be operatively connected with one or more enzymes for the enzymatic route that generates the non-alienation compound of allos and for driving it to express.In some embodiments, host cell comprises the nucleic acid of coding GAL4 further.In some embodiments, GAL4 gene product constructive expression, namely under composition promotor controls.In some embodiments, host cell is included in the nucleic acid of the coding GAL80 under maltose responsiveness promotor described herein control further, and induces GAL80 gene product expression under maltose exists.Gal80p then interacts with Gal4p and stops Gal4p transcriptional activity.Remove when maltose or fully consume, when making no longer to induce GAL80 to express, Gal4p removes preventing of Gal80p, and freely activates one or more expression of enzymes of the enzymatic route for generating the non-alienation compound of allos.

In other embodiments, the heterologous nucleic acids of natural pGAL4 promotor involved maltose responsiveness promotor is replaced.In some embodiments, host cell comprises heterologous nucleic acids, and it comprises the nucleic acid of coding Gal4p, is operatively connected with the heterologous nucleic acids comprising maltose responsiveness promotor.In one embodiment, the encoding sequence of maltose responsiveness promotor and Gal4p is operatively connected, and be operatively connected for the encoding sequence of one or more enzymes (such as all members) of the enzymatic route that generates allos non-alienation compound and GAL4 responsiveness promotor, make to induce one or more expression of enzymes under maltose exists.In some embodiments, GAL4 responsiveness promotor is pGAL1.In some embodiments, GAL4 responsiveness promotor is pGAL7.In some embodiments, GAL4 responsiveness promotor is pGAL10.

Preventing and the non-amount of preventing of 3.2 maltose

As follows, maltose is the disaccharides formed from 2 glucose molecules.It has chemical formula C ₁₂h ₂₂o ₁₁, and molecular weight 343g/mol.

(being namely enough to the maltose amount that encoding sequence that induction and maltose responsiveness promotor be operatively connected is expressed) in some embodiments of the method provided in this article, can be measured herein for " induction " of the maltose in the method provided for any genetically modified host cell that can generate allos non-alienation compound measures and " non-induced " of maltose is measured (namely lower than the maltose amount that it does not then induce the encoding sequence be operatively connected with maltose responsiveness promotor to express).In some embodiments, the non-induced amount of maltose implements Gene expression profiles under the maltose by there is increasing amounts in the substratum that will be used for zymotechnique, and namely maltose titration measures.Such as, host cell genetically modified for a group can be divided into multiple subgroup and parallelly cultivate, wherein in the substratum comprising the maltose (comprising without maltose) that difference (such as cumulative) is measured, cultivate each subgroup, and after limiting time section, measure reporter gene expression or the generation of non-alienation compound.

Be routed in maltose responsiveness promotor in some embodiments realizing non-alienation compound generation "Off" state under maltose exists, maltose titration comprises at least two maltose concentrations, the non-alienation compound of host cell generates and reaches minimum platform thus, does not namely observe compound generation reduction further along with maltose concentration raises.In some embodiments, " preventing " amount of maltose is at least the maltose minimum that the non-alienation compound of host cell generates when reaching its minimum platform (such as zero).This amount can also be called maltose " saturated " for preventing non-alienation compound to generate to particular host cell or " the best " amount.In some these type of embodiments, " preventing " amount of maltose can comprise non-alienation compound and generate relative to "On" state reduction, even has any maltose concentration that low-level compound generates.In some embodiments, in this structure of switch, the non-amount of preventing of maltose " prevents " lower than maltose any maltose amount measured.In some embodiments, maltose the non-amount of preventing than maltose the amount of preventing little at least 2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100 or be greater than 100 times.In one particular embodiment, the non-amount of preventing of maltose is less than 0.8% (w/v) of substratum.In another particular, the non-amount of preventing of maltose is less than 0% (w/v) of substratum.

In a specific embodiment, as mentioned above, the amount of preventing of maltose is best or saturation capacity for given host cell, but not the amount of preventing is without maltose.In another embodiment, the amount of preventing of maltose is at least 0.25%, but not the amount of preventing is without maltose.In another embodiment, the amount of preventing of maltose is the maltose amount of 0.25% to 3%, but not the amount of preventing is without maltose.In another embodiment, the amount of preventing of maltose is at least 3%, and limited volume is without maltose.

Be routed in some embodiments realizing non-alienation compound generation "Off" state under maltose exists in maltose responsiveness promotor, in substratum, the amount of preventing of maltose is at least 0.1% (the every volume medium of weight maltose).In some embodiments, in substratum, the amount of preventing of maltose is at least 0.25%.In some embodiments, in substratum, the amount of preventing of maltose is at least 0.5%.In some embodiments, in substratum, the amount of preventing of maltose is at least 0.75%.In some embodiments, in substratum, the amount of preventing of maltose is at least 1.0%.In some embodiments, in substratum, the amount of preventing of maltose is at least 1.25%.In some embodiments, in substratum, the amount of preventing of maltose is at least 1.5%.In some embodiments, in substratum, the amount of preventing of maltose is at least 1.75%.In some embodiments, in substratum, the amount of preventing of maltose is at least 2.0%.In some embodiments, in substratum, the amount of preventing of maltose is at least 2.25%.In some embodiments, in substratum, the amount of preventing of maltose is at least 2.5%.In some embodiments, in substratum, the amount of preventing of maltose is at least 2.75%.In some embodiments, in substratum, the amount of preventing of maltose is at least 3.0%.In some embodiments, in substratum, the amount of preventing of maltose is at least 3.25%.In some embodiments, in substratum, the amount of preventing of maltose is at least 3.5%.In some embodiments, in substratum, the amount of preventing of maltose is at least 3.75%.In some embodiments, in substratum, the amount of preventing of maltose is at least 4.0%.In some embodiments, in substratum, the amount of preventing of maltose is at least 4.25%.In some embodiments, in substratum, the amount of preventing of maltose is at least 4.5%.In some embodiments, in substratum, the amount of preventing of maltose is at least 4.75%.In some embodiments, in substratum, the amount of preventing of maltose is at least 5.0%.In some embodiments, in substratum, the amount of preventing of maltose is between 5% and 50%.In some embodiments, in substratum, the amount of preventing of maltose is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or about 50%.

In some embodiments, the non-amount of preventing of maltose is less than the amount of preventing of the maltose measured according to method mentioned above at least 2 times, 10 times, 100 times, 1000 times, the amount of 10,000 times or 100,000 times.In some embodiments, the non-amount of preventing of maltose is less than the saturation capacity of the maltose measured according to method mentioned above at least 2 times, 10 times, 100 times, 1000 times, the amount of 10,000 times or 100,000 times.In some embodiments, the non-amount of preventing of maltose be the amount of preventing of the maltose measured according to method mentioned above be less than 50%, be less than 20%, be less than 10%, be less than 1%, be less than 0.5%, be less than 0.2%, be less than 0.1%, be less than 0.01%, or be less than the amount of 0.001%.In some embodiments, the non-amount of preventing of maltose be the saturation capacity of the maltose measured according to method mentioned above be less than 50%, be less than 20%, be less than 10%, be less than 1%, be less than 0.1%, be less than 0.01%, or be less than the amount of 0.001%.In a specific embodiment, the non-amount of preventing of maltose is 0mg/L (0%), namely without maltose.So, in this specific embodiment, during the production phase, in not containing the cell culture medium in outside maltose source, host cell is cultivated.

Be routed in maltose responsiveness promotor in some embodiments realizing non-alienation compound generation "On" state under maltose exists, maltose titration comprises at least two maltose concentrations, the non-alienation compound of host cell generates and reaches maximum platform thus, does not namely observe compound generation rising further along with maltose concentration raises.In some embodiments, " non-prevent " amount of maltose is at least the maltose minimum that the non-alienation compound of host cell generates when reaching its maximum platform.In this structure of switch, this amount can also be called maltose " saturated " for inducing non-alienation compound to generate to particular host cell or " the best " amount.In some these type of embodiments, " induction " amount of maltose can comprise non-alienation compound and generate relative to "Off" state rising, and even compound generation is any maltose concentration of suboptimal.In some embodiments, in this structure of switch, the non-induced amount of maltose is any maltose amount measured lower than maltose " induction ".In some embodiments, maltose non-induced amount than maltose inducing amount little at least 2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100 or be greater than 100 times.In one particular embodiment, the non-induced amount of maltose is less than 0.8% (w/v) of substratum.In another particular, the non-induced amount of maltose is less than 0% (w/v) of substratum.

In a specific embodiment, as mentioned above, the inducing amount of maltose is best or saturation capacity to given host cell, but not inducing amount is without maltose.In another embodiment, the inducing amount of maltose is at least 0.25%, but not inducing amount is without maltose.In another embodiment, the inducing amount of maltose is the maltose amount of 0.25% to 3%, but not inducing amount is without maltose.In another embodiment, the inducing amount of maltose is at least 3%, and limited volume is without maltose.

Be routed in some embodiments realizing non-alienation compound generation "On" state under maltose exists in maltose responsiveness promotor, in substratum, the inducing amount of maltose is at least 0.1% (the every volume medium of weight maltose).In some embodiments, in substratum, the inducing amount of maltose is at least 0.25%.In some embodiments, in substratum, the inducing amount of maltose is at least 0.5%.In some embodiments, in substratum, the inducing amount of maltose is at least 0.75%.In some embodiments, in substratum, the inducing amount of maltose is at least 1.0%.In some embodiments, in substratum, the inducing amount of maltose is at least 1.25%.In some embodiments, in substratum, the inducing amount of maltose is at least 1.5%.In some embodiments, in substratum, the inducing amount of maltose is at least 1.75%.In some embodiments, in substratum, the inducing amount of maltose is at least 2.0%.In some embodiments, in substratum, the inducing amount of maltose is at least 2.25%.In some embodiments, in substratum, the inducing amount of maltose is at least 2.5%.In some embodiments, in substratum, the inducing amount of maltose is at least 2.75%.In some embodiments, in substratum, the inducing amount of maltose is at least 3.0%.In some embodiments, in substratum, the inducing amount of maltose is at least 3.25%.In some embodiments, in substratum, the inducing amount of maltose is at least 3.5%.In some embodiments, in substratum, the inducing amount of maltose is at least 3.75%.In some embodiments, in substratum, the inducing amount of maltose is at least 4.0%.In some embodiments, in substratum, the inducing amount of maltose is at least 4.25%.In some embodiments, in substratum, the inducing amount of maltose is at least 4.5%.In some embodiments, in substratum, the inducing amount of maltose is at least 4.75%.In some embodiments, in substratum, the inducing amount of maltose is at least 5.0%.In some embodiments, in substratum, the inducing amount of maltose is between 5% and 50%.In some embodiments, in substratum, the inducing amount of maltose is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%45% or about 50%.

In some embodiments, the non-amount of preventing of maltose is less than the amount of preventing of the maltose measured according to method mentioned above at least 2 times, 10 times, 100 times, 1000 times, the amount of 10,000 times or 100,000 times.In some embodiments, the non-amount of preventing of maltose is less than the saturation capacity of the maltose measured according to method mentioned above at least 2 times, 10 times, 100 times, 1000 times, the amount of 10,000 times or 100,000 times.In some embodiments, the non-amount of preventing of maltose be the amount of preventing of the maltose measured according to method mentioned above be less than 50%, be less than 20%, be less than 10%, be less than 1%, be less than 0.5%, be less than 0.2%, be less than 0.1%, be less than 0.01%, or be less than the amount of 0.001%.In some embodiments, the non-amount of preventing of maltose be the saturation capacity of the maltose measured according to method mentioned above be less than 50%, be less than 20%, be less than 10%, be less than 1%, be less than 0.1%, be less than 0.01%, or be less than the amount of 0.001%.In a specific embodiment, the non-amount of preventing of maltose is 0mg/L (0%), namely without maltose.So, in this specific embodiment, in not containing the cell culture medium in outside maltose source, during the production phase, host cell is cultivated.

The generation of 3.3 non-catabolites

In some embodiments of the fermentation process provided in this article, utilize and the micro-aerobic responsiveness promotor of oxygen condition operative combination or the maltose responsiveness promotor that combines with maltose conditional operation, the non-alienation compound of period construction phase (step (a) of method mentioned above) generates 50 of the maximum non-alienation compound generation (such as when the amount that the non-alienation compound when period production phase (step (b) of method mentioned above) cultivates host cell generates) being less than genetically modified host cell, 40,30,20 or 10%.

The time period of construction phase and production phase that period carries out zymotechnique can change, and can depend on the growth velocity of such as host cell, the factors such as the inherent growth velocity of host cell; And other culture condition, such as pH, temperature, this depends on host cell, fermentation, and the specific requirement of technique.But the construction phase expection of any time length produces some benefits to final fermentation productivity, is removed in "Off" state because a certain amount of and non-alienation compound generates relevant negative selective pressure.

In some embodiments, the construction phase carries out for some time of being enough to generate a certain amount of cellular biomass (it can support during the production phase that non-alienation compound generates).In some embodiments, construction phase group's experience of carrying out existing when being enough to make inoculation repeatedly doubly increases to for some time of the cell density reaching expectation.In some embodiments, the host cell group carried out in the fermentation vessel that are enough to make to carry out the construction phase or container reaches the cell density (OD between 0.01 and 400 construction phase ₆₀₀) for some time.In some embodiments, the construction phase proceeds to the OD reaching at least 0.01 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reaching at least 0.1 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reaching at least 1.0 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reaching at least 10 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reaching at least 100 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reached between 0.01 and 100 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reached between 0.1 and 10 ₆₀₀.In some embodiments, the construction phase proceeds to the OD reached between 1 and 100 ₆₀₀.In other embodiments, the construction phase carries out one section at least 12,24, and 36,48,60,72,84,96 or more than 96 hours.

In some embodiments, the production phase carries out for some time of the non-alienation compound being enough to generation desired amount.In some embodiments, the production phase carries out one section at least 12,24, and 36,48,60,72,84,96 or more than 96 hours.In some embodiments, the production phase carried out one section between 3 and 20 days.In some embodiments, the production phase carries out one section 1, and 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more than 20 days.

In one particular embodiment, the method generating non-alienation compound carries out the fermentation of genetically modified host cell under being included in and being enough to allow the aerobic condition of genetically modified host cell growth and maintenance; Then the micro-aerobic fermentation condition being enough to induce non-alienation compound to generate is provided subsequently, and runs through the micro-aerobic condition of fermentation operation maintenance.

In another embodiment, the method generating non-alienation compound is included in construction separately and generates in substratum cultivates host cell.Such as, method can be included in the construction phase and cultivate genetically modified host cell, wherein under non-formation condition culturing cell to generate inoculum, then under the condition being suitable for inducing compounds generation, inoculum is transferred to the second fermention medium, and in the second fermentation stage, maintains steady state conditions to generate the cell culture containing non-catabolite.

In some embodiments, the amount that the method provided herein is enough to be greater than about 10 grams of often liter of fermention mediums generates one or more non-alienation compounds.In some these type of embodiments, non-alienation derivative compound is with about 10 to about 50 grams, exceedes about 15 grams, exceedes about 20 grams, exceed about 25 grams, or exceed about 30 grams of often liter of cell cultures amount generate.

In some embodiments, the amount that the method provided herein is enough to be greater than about 50 milligrams of every gram of dry cell weights generates one or more non-alienation compounds.In some embodiments, the non-alienation compound that restructuring generates is with about 50 to about 1500 milligrams, exceed about 100 milligrams, exceed about 150 milligrams, exceed about 200 milligrams, exceed about 250 milligrams, exceed about 500 milligrams, exceed about 750 milligrams, or exceed about 1000 milligrams of every gram of dry cell weights amount generate.

In some embodiments, the non-alienation compound that the enforcement of the method provided herein causes host cell group genetically modified compared with generating caused by the method not containing the construction phase (period cultivates host cell under non-formation condition) to raise generates.In some embodiments, the enforcement of method causes with higher at least about 10% than the amount of the non-alienation compound by not generating containing the method for construction phase (period cultivates host cell under non-formation condition), at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1, 000 times, or more amount generate one or more non-alienation compounds, based on the cell culture of per unit volume.

In some embodiments, the enforcement of method causes with higher at least about 10% than the amount of the non-alienation compound by not generating containing the method for construction phase (period cultivates host cell under non-formation condition), at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1, 000 times, or more amount generate one or more non-alienation compounds, based on per unit dry cell weight.

In some embodiments, the enforcement of method causes with higher at least about 10% than the amount of the non-alienation compound by not generating containing the method for construction phase (period cultivates host cell under non-formation condition), at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1, 000 times, or more amount generate one or more non-alienation compounds, based on the cell culture time per unit of per unit volume.

In some embodiments, the enforcement of method causes with higher at least about 10% than the amount of the non-alienation compound by not generating containing the method for construction phase (period cultivates host cell under non-formation condition), at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1, 000 times, or more amount generate one or more non-alienation compounds, based on per unit dry cell weight time per unit.

3.4 substratum and culture condition

The materials and methods maintained for microorganisms cultures and grow is that microbiology or fermentation science those skilled in the art are known (see such as Bailey et al., Biochemical EngineeringFundamentals, second edition, McGraw Hill, New York, 1986).According to host cell, fermentation, and the specific requirement of technique, must to appropriate media, pH, temperature, and to aerobic, micro-aerobic, or the requirement of anaerobic condition is considered.

The method of the non-alienation compound of generation that can implement to provide herein in suitable vessel in suitable culture medium, includes but not limited to Tissue Culture Plate, flask, or fermentor tank.Further, can with the industrial any fermentation-scale implementation method of support microbial product known in the art.Any suitable fermentor tank can be used, comprise and stir tank fermentor tank, airlift fermentor, bubble fermentation tank, or its any combination.Utilizing yeast saccharomyces cerevisiae as in the particular of host cell, bacterial strain can be cultivated in fermentor tank, as Kosaric, et al, in Ullmann's Encyclopedia of Industrial Chemistry, SixthEdition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co.KDaA, Weinheim, Germany record in detail.Further, with any fermentation volume implementation method, such as, can ferment to technical scale (such as 500L is to >=500,000L) to pilot scale (such as 20L to 500L) from laboratory scale (such as 10ml to 20L).

In some embodiments, comprise the genetically modified microbe that can generate non-alienation compound for the substratum in the method for the generation provided non-alienation compound herein can survive wherein, namely support and maintain any substratum of growth and viability.In some embodiments, substratum also promotes to generate the biosynthetic pathway expecting that non-alienation compound is required.

In some embodiments, substratum for comprising assimilable carbon, the water-containing medium of nitrogen and source of phosphoric acid.This type of substratum can also comprise suitable salt, mineral, metal and other nutrition.In some embodiments, incrementally or continuously carbon source and each must be added into fermention medium by cytotrophy thing, and often kind of nutrition cell be maintained in growth needed effectively assimilates the minimum level substantially of needs, such as, carbon source is transformed into the metabolism of biomass or respiratory function according to the cell growth curve measured in advance based on cell.

Well known in the art for the conditions suitable of culturing micro-organisms body and suitable culture medium.In some embodiments, one or more other reagent is supplemented to suitable culture medium, such as such as inductor (such as when the nucleotides sequence of one or more encodes gene product is listed under inducible promoters control), repressor (such as when the nucleotides sequence of one or more encodes gene product be listed in can repressible promoter control under time), or selective agent (such as selecting the biocide of the microbe comprising genetic modification).

In some embodiments, carbon source is monose (simply sugar), disaccharides, polysaccharide, not fermentable carbon source, or one or more its combination.The non-limiting examples of suitable monose comprises glucose, semi-lactosi, seminose, fructose, ribose, and combination.The non-limiting examples of suitable disaccharides comprises sucrose, lactose, maltose, trehalose, cellobiose, and combination.The non-limiting examples of Suitable polysaccharides comprises starch, glycogen, Mierocrystalline cellulose, chitin, and combination.The non-limiting examples of suitable not fermentable carbon source comprises acetate and glycerine.

In substratum, the concentration of carbon source (such as glucose) should Promote cell's growth, but Ying Gaozhi does not prevent the microbe of use to grow.Typically, run with carbon source (such as glucose) and cultivate, the level of interpolation realizes growth and the biomass of aspiration level, but can't detect (detection is limited to about <0.1g/l).In other embodiments, in substratum, the concentration of carbon source (such as glucose) is greater than about 1g/L, is preferably greater than about 2g/L, and more preferably greater than about 5g/L.In addition, in substratum, the concentration typical case of carbon source (such as glucose) is less than about 100g/L, is preferably less than about 50g/L, and is more preferably less than about 20g/L.It should be noted that to mention and cultivate constituent concentration and can refer to initially and/or both constituent concentrations at that time.In some cases, may expect that allowing substratum to become carbon source exhausts in the training period.

The assimilable nitrogen source that can use in suitable culture medium includes but not limited to simple nitrogenous source, organic nitrogen source and complicated nitrogenous source.This type of nitrogenous source comprises anhydrous ammonia, ammonium salt and animal, the material of plant and/or microbial origin.Suitable nitrogenous source includes but not limited to protein hydrolystate, microbial biomass hydrolyzate, peptone, yeast extract, ammonium sulfate, urea, and amino acid.Typically, in substratum, the concentration of nitrogenous source is greater than about 0.1g/L, is preferably greater than about 0.25g/L, and more preferably greater than about 1.0g/L.But, exceed some concentration interpolation nitrogenous source not favourable to microbe growth to substratum.As a result, the concentration of nitrogenous source is less than about 20g/L in substratum, is preferably less than about 10g/L and is more preferably less than about 5g/L.Further, in some cases, may expect that allowing substratum to become nitrogenous source exhausts in the training period.

Effective culture medium is had to contain other compound, such as inorganic salt, VITAMIN, trace-metal or growth stimulant.This type of other compound also can be present in the carbon had in effective culture medium, in nitrogen or mineral sources or can be added into substratum specially.Substratum can also contain suitable phosphoric acids source.This type of source of phosphoric acid comprises inorganic and both organophosphorus acid sources.Preferred source of phosphoric acid includes but not limited to phosphoric acid salt such as list or Disodium Hydrogen Phosphate and potassium, ammonium phosphate and composition thereof.Typically, in substratum, the concentration of phosphoric acid is greater than about 1.0g/L, is preferably greater than about 2.0g/L and more preferably greater than about 5.0g/L.But, exceed some concentration interpolation phosphoric acid not favourable to microbe growth to substratum.Thus, in substratum, the concentration typical case of phosphoric acid is less than about 20g/L, is preferably less than about 15g/L and is more preferably less than about 10g/L.

Suitable culture medium can also comprise magnesium source, is preferably in the form of physiology acceptable salt, such as magnesium sulfate 7 hydrate, although can use other magnesium source with the concentration of the magnesium contributing analog quantity.Typically, in substratum, the concentration of magnesium is greater than about 0.5g/L, is preferably greater than about 1.0g/L, and more preferably greater than about 2.0g/L.But, exceed some concentration interpolation magnesium not favourable to microbe growth to substratum.Thus, in substratum, the concentration typical case of magnesium is less than about 10g/L, is preferably less than about 5g/L, and is more preferably less than about 3g/L.Further, in some cases, may expect that allowing substratum to become magnesium source exhausts in the training period.

In some embodiments, substratum can also comprise biology acceptable chelating agent, such as citrate trisodium dihydrate.In this type of situation, in substratum, the concentration of sequestrant is greater than about 0.2g/L, is preferably greater than about 0.5g/L, and more preferably greater than about 1g/L.But, exceed some concentration interpolation sequestrant not favourable to microbe growth to substratum.Thus, in substratum, the concentration typical case of sequestrant is less than about 10g/L, is preferably less than about 5g/L, and is more preferably less than about 2g/L.

Substratum can also comprise biology at first can accept acid or alkali with the expectation pH of maintain base.Biology can accept acid and include but not limited to hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and composition thereof.Biology can accept alkali and include but not limited to ammonium hydroxide, sodium hydroxide, potassium hydroxide and composition thereof.In some embodiments, the alkali of use is ammonium hydroxide.

Substratum can also comprise biology can accept calcium source, includes but not limited to calcium chloride.Typically, in substratum, the concentration of calcium source (such as calcium chloride dihydrate) is in the scope of about 5mg/L to about 2000mg/L, preferably in the scope of about 20mg/L to about 1000mg/L, and more preferably in the scope of about 50mg/L to about 500mg/L.

Substratum can also comprise sodium-chlor.Typically, in substratum, the concentration of sodium-chlor is in the scope of about 0.1g/L to about 5g/L, preferably in the scope of about 1g/L to about 4g/L, and more preferably in the scope of about 2g/L to about 4g/L.

In some embodiments, substratum can also comprise trace-metal.This type of trace-metal can be added into substratum with the form of liquid storage, and conveniently, liquid storage can separate with the rest part of substratum and prepare.Typically, the amount being added into this type of trace metal solutions of substratum is greater than about 1ml/L, is preferably greater than about 5mL/L, and more preferably greater than about 10mL/L.But, exceed some concentration interpolation trace-metal not favourable to microbe growth to substratum.Thus, the amount typical case being added into this type of trace metal solutions of substratum is less than about 100mL/L, is preferably less than about 50mL/L, and is more preferably less than about 30mL/L.It should be noted that adding outside trace-metal with liquid storage, can separately add various composition, in each comfortable respective range, no matter the amount of composition that specifies of trace metal solutions scope above.

Substratum can comprise other VITAMIN, such as vitamin H, calcium, pantothenate, inositol, pyridoxol-HCl, and thiamines-HCl.This biostearin can be added into substratum with the form of liquid storage, and conveniently, liquid storage can separate with the rest part of substratum and prepare.But, exceed some concentration interpolation VITAMIN not favourable to microbe growth to substratum.

Fermentation process described herein can be implemented with cellar culture pattern, includes but not limited in batches, fedbatch, cell recirculation, continuously with semicontinuous.In some embodiments, ferment with Fed-batch mode.In this type of situation, exhaust between some compositions of substratum incubation period during the production phase of fermentation.In some embodiments, the specific examples of such components of relatively high density can be compensated to culture when such as the production phase starts, make before needs add, support that growth and/or non-alienation compound generate for some time.By adding with the level of culture consumption, run through the preferable range of cultivating and maintaining these compositions.Substratum sampled by such as regular and measure concentration, the level of each composition in substratum can be monitored.Or, once develop type culture code, just can run through to cultivate and add with the fixed time interval corresponding with known level at specified time.Can be familiar with as those skilled in the art, nutrition wear rate raises with the cell density rising of substratum in the training period.In addition, in order to avoid external microbe is introduced substratum, aseptic addition means known in the art is used to implement to add.In addition, a small amount of defoamer can be added in the training period.

The temperature of substratum can be any temperature being suitable for genetically modified Growth of Cells and/or the generation of non-alienation compound.Such as, before giving culture medium inoculated inoculum, substratum can be taken to and be maintained at the temperature in the scope of about 20 DEG C to about 45 DEG C, the temperature preferably in the scope of about 25 DEG C to about 40 DEG C, and more preferably in the scope of about 28 DEG C to about 32 DEG C.

The pH of substratum can by adding acid or alkali controls to substratum.In this type of situation, when using ammoniacal liquor control pH, it also serves as the nitrogenous source in substratum easily.Preferably, pH is maintained at about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.

In some embodiments, the carbon source concentration (such as maltose or glucose concn) of substratum is monitored in the training period.The glucose concn of substratum can use known technology to monitor, such as such as use glucose oxidase test or high pressure liquid chromatography, they can be used in monitoring the glucose concn in supernatant liquor (such as cell-free medium composition), and maltose level can be monitored similarly.As discussed previously, carbon source concentration should keep below and cytostatic level occurs.Although this type of concentration can change between organism, for glucose as carbon source, the glucose concn that cell growth inhibition is being greater than about 60g/L occurs, and easily can be measured by test.Thus, when use glucose as carbon source time, preferably by glucose feed supplement to fermentor tank and maintain lower than detectability.Or the glucose concn in substratum maintains in the scope of about 1g/L to about 100g/L, more preferably in the scope of about 2g/L to about 50g/L, and still more preferably in the scope of about 5g/L to about 20g/L.Although carbon source concentration can by adding such as substantially pure glucose solution and maintain in aspiration level, can to accept and may preferably by the carbon source concentration adding aliquot initial medium and come maintain base.May expect to use aliquot initial medium, because can the concentration of other nutrition (such as nitrogen and source of phosphoric acid) in maintain base simultaneously.Similarly, the trace metal concentrations in substratum can maintain by adding aliquot trace metal solutions.

The recovery of 3.5 non-alienation compounds

Once generate non-alienation compound by host cell, any appropriate separation known in the art and purification process just can be used to carry out reclaiming or being separated for follow-up.In some embodiments, by centrifugal, the organic phase comprising non-alienation is separated with fermented liquid.In other embodiments, the organic phase comprising non-alienation compound is spontaneously separated with fermented liquid.In other embodiments, by emulsion splitter and/or nucleator are added into fermentation reaction, the organic phase comprising non-alienation derivative compound is separated with fermented liquid.The illustrative example of emulsion splitter comprises flocculation agent and condensing agent.The illustrative example of nucleator comprises the droplet of non-alienation compound self and organic solvent (such as dodecane, Isopropyl myristate, and Witconol 2301).

The non-alienation compound that generates in these cells may reside in culture supernatants neutralization/or associates with host cell.In the embodiment that non-alienation compound associates with host cell, the recovery of non-alienation compound can the method for comprising or lysing cell.Or/simultaneously, recovery process can be used to reclaim the non-alienation compound in substratum, include but not limited to chromatography, extraction, solvent extraction, membrane sepn, electrodialysis, reverse osmosis, distillation, chemically derived and crystallization.

In some embodiments, non-alienation compound and other product that can be present in organic phase are separated.In some embodiments, be separately use absorption, distillation, solution-air extraction (stripping), liquid-liquid extraction (solvent extraction), ultrafiltration, and Standard chromatographic techniques realization.

In some embodiments, the non-alienation compound of recovery is pure, such as pure at least about 40%, pure at least about 50%, pure at least about 60%, pure at least about 70%, pure at least about 80%, pure at least about 90%, pure at least about 95%, pure at least about 98%, or it is pure more than 98%, wherein " pure " does not contain other non-alienation compound, pollutent, the non-alienation compound waited at the linguistic context middle finger of non-alienation compound.

4. genetically modified microbe

Provide the genetically modified microbe (such as genetically modified brewing yeast cell) generating derivative (non-alienation) compound of allos acetyl-CoA herein.Compared with lacking the parental microorganism body of genetic modification described herein, genetically modified microbe generate more substantial one or more from the biosynthetic compound of acetyl-CoA.

The method using expression vector or chromosomal integration construction genetically modified microorganism (one or more the non-alienation compounds such as in order to realize raising in host cell generate) is well known in the art.See such as Sherman, F., et al., Methods Yeast Genetics, Cold Spring HarborLaboratory, N.Y. (1978); Guthrie, C., et al. (Eds.) Guide To Yeast Genetics andMolecular Biology Vol.194, Academic Press, San Diego (1991); Sambrook et al., 2001, Molecular Cloning--A Laboratory Manual, 3 ^rdedition, Cold SpringHarbor Laboratory, Cold Spring Harbor, NY; And Ausubel et al., eds., CurrentEdition, Current Protocols in Molecular Biology, Greene Publishing Associatesand Wiley Interscience, NY.; By addressing by its disclosure income herein.In addition, by deleting, sudden change, and/or gene rearrangement realizes gene expression inhibition, such as this causes one or more the non-alienation compounds raised in cell to generate.It also can use sense-rna, siRNA, miRNA, ribozyme, triple strand dna, and transcribes and/or translational inhibitor is carried out.In addition, transposon can be adopted to destroy genetic expression, such as, by it being inserted between promotor and coding region, or with deactivation two kinds of genes one or both of between two neighboring gene.

In some embodiments, the non-alienation compound generation realizing raising in cell by using expression vector to express specific protein (such as involving the protein of biosynthetic pathway mentioned above).Usually, expression vector is the recombination of polynucleotide molecule comprising reproducing signals and the expression control sequenc (such as promotor and terminator) be operatively connected with the nucleotide sequence of coded polypeptide.Virus vector (such as retrovirus, adenovirus and adeno associated virus) is comprised, plasmid vector, and clay to the expression vector of expressing polypeptide-coding nucleotide sequence useful.The illustrative example being adapted at the expression vector used in yeast cell includes but not limited to CEN/ARS and 2 μ plasmids.The illustrative example being suitably in the promotor used in yeast cell includes but not limited to the promotor of the TEF1 gene of K.lactis, the promotor of the PGK1 gene of yeast saccharomyces cerevisiae, the promotor of the TDH3 gene of yeast saccharomyces cerevisiae, can repressible promoter, the promotor of the CTR3 gene of such as yeast saccharomyces cerevisiae, and inducible promoters, galactose-inducible promoter (the such as GAL1 of such as yeast saccharomyces cerevisiae, GAL7, and the promotor of GAL10 gene).

By any method that those skilled in the art will know that, expression vector and chromosomal integration construction can be introduced microorganism cells ad lib.See such as Hinnen et al., Proc.Natl.Acad.Sci.USA 75:1292-3 (1978); Cregg et al., Mol.Cell.Biol.5:3376-3385 (1985); U.S. Patent No. 5,272,065; Goeddel et al., eds, 1990, Methods in Enzymology, vol.185, Academic Press, Inc., CA; Krieger, 1990, Gene Transfer and Expression--ALaboratory Manual, Stockton Press, NY; Sambrook et al., 1989, MolecularCloning--A Laboratory Manual, Cold Spring Harbor Laboratory, NY; And Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.Illustrative technique includes but not limited to spheroplast (spheroplasting), electroporation, the conversion that PEG 1000 mediates, and the conversion of Lithium Acetate or lithium chloride mediation.

4.1 host cell

Cell useful in the method and composition provided in this article comprises and natural or restructuring can generate any cell of non-alienation compound (such as isoprenoid, polyketone, lipid acid, like this).In some embodiments, cell is prokaryotic cell prokaryocyte.In some embodiments, cell is bacterial cell.In some embodiments, cell is Bacillus coli cells.In some embodiments, cell is eukaryotic cell.In some embodiments, cell is mammalian cell.In some embodiments, cell is Chinese hamster ovary (CHO) cell, COS-7 cell, l cell, mice embryonic cancer cells, or mouse embryo stem cell.In some embodiments, cell is insect cell.In some embodiments, cell is S2 cell, Schneider cell, S12 cell, 5B1-4 cell, Tn5 cell, or Sf9 cell.In some embodiments, cell is unicellular eukaryote somatocyte.

In some embodiments, cell is filamentous bacteria cells.In some embodiments, filamentous bacteria cells is actinomycetes (actinomycete) guiding principle.In specific embodiments, filamentous bacteria cells is that streptomycete (Streptomyces) belongs to, such as produce dyadic streptomycete (Streptomyces ambofaciens), deinsectization streptomycete (Streptomyces avermitilis), blue or green streptomycete far away (Streptomyces azureus), Chinese cassia tree streptomycete (Streptomyces cinnamonensis), streptomyces coelicolor (Streptomycescoelicolor), Gu Lake streptomycete (Streptomyces curacoi), Streptomyces erythraeus, streptomyces fradiae (Streptomyces fradiae), Galilee streptomycete (Streptomyces galilaeus), Streptomyces glaucescens (Streptomyces glaucescens), streptomyces hygroscopicus (Streptomyceshygroscopicus), muta lead mycillin (Streptomyces lividans), small streptomycete (Streptomyces parvulus), ripple plug streptomycete (Streptomyces peucetius), streptomyces rimosus (Streptomyces rimosus), rose Streptomyces fulvissimus (Streptomyces roseofulvus), heat-resisting streptomycete (Streptomyces thermotolerans), Streptomyces violaceoruber (Streptomycesviolaceoruber).

In another embodiment, cell is fungal cell.In one particularly embodiment, cell is yeast cell.Yeast useful in the method and composition provided in this article comprises and is preserved in microbial preservation mechanism (such as IFO, ATCC, Deng) and belong to following genus yeast: pin gemma yeast (Aciculoconidium), god's food yeast (Ambrosiozyma), condyle yeast (Arthroascus), Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces (Brettanomyces), Bu Le bullet spore yeast (Bullera), Bulleromyces, candiyeast (Candida), Citeromycesbaodingensis (Citeromyces), rod spore yeast (Clavispora), Cryptococcus (Cryptococcus), Cystofilobasidium, Dbaly yeast (Debaryomyces), moral gram yeast (Dekkara), Dipodascopsis, Dipodascus, Ai Nila yeast (Eeniella), endomycopsi.sp (Endomycopsella), Eremascus, Eremothecium, Erythrobasidium, Fellomyces, line ustilago (Filobasidium), Galactomyces, ground mould (Geotrichum), little season yeast (Guilliermondella), there is spore debaryomyces hansenii (Hanseniaspora), Hansenula, Hasegawaea, Holtermannia, chain yeast (Hormoascus), Hyphopichia, her Sa yeast (Issatchenkia), Ke Leke yeast (Kloeckera), Kloeckeraspora, kluyveromyces (Kluyveromyces), Kondoa, Kuraishia, Kurtzmanomyces, white winter spore yeast (Leucosporidium), saccharomyces oleaginosus (Lipomyces), class moral yeast (Lodderomyces), Malassezia, the strange yeast of enzyme (Metschnikowia), Mrakia, Myxozyma, receive inferior yeast (Nadsonia), Nakazawaea, pin spore yeast (Nematospora), Ogataea, ovum spore yeast (Oosporidium), pipe capsule yeast (Pachysolen), heavy wall spore yeast (Phachytichospora), Fan Feiya yeast (Phaffia), pichia spp (Pichia), red winter spore yeast (Rhodosporidium), rhodotorula (Rhodotorula), (sugar) yeast (Saccharomyces), class yeast (Saccharomycodes), multiple film spore yeast (Saccharomycopsis), Saitoella, Sakaguchia, Saturnospora, split budding yeast (Schizoblastosporion), fragmentation (sugar) yeast (Schizosaccharomyces), permitted Wang Shi yeast (Schwanniomyces), sealed shadow yeast (Sporidiobolus), shadow yeast (Sporobolomyces), spore pachyderma yeast (Sporopachydermia), hat capsule yeast (Stephanoascus), stalk spore yeast (Sterigmatomyces), intend stalk spore yeast (Sterigmatosporidium), Symbiotaphrina, sympodium yeast (Sympodiomyces), Sympodiomycopsis, there is spore torula (Torulaspora), Trichosporiella, trichosporon (Trichosporon), trigonopsis variabilis (Trigonopsis), Tsuchiyaea, Udeniomyces, Waltomyces, Brunswick yeast (Wickerhamia), intend Brunswick yeast (Wickerhamiella), intend Weir yeast (Williopsis), Yamadazyma, Yarrowia sp (Yarrowia), zygosaccharomyces (Zygoascus), Zygosaccharomyces, Zygowilliopsis and Zygozyma etc.

In specific embodiments, yeast useful in the method and composition provided in this article comprises yeast saccharomyces cerevisiae, pichia pastoris phaff (Pichia pastoris), schizosaccharomyces pombe (Schizosaccharomyces pombe), Dekkera bruxellensis, Kluyveromyces lactis (Kluyveromyces lactis) (being called lactic acid sugar yeast (Saccharomyces lactis) in the past), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorpha (Hansenulapolymorpha) (being called now Pichia angusta).In some embodiments, microorganism is mycocandida (such as Candida lipolytica (Candida lipolytica), Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis (Candida utilis)) bacterial strain.

In one particular embodiment, cell is brewing yeast cell.In some embodiments, the bacterial strain of brewing yeast cell is selected from lower group: bread yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1.In some embodiments, the bacterial strain of yeast saccharomyces cerevisiae is selected from lower group: PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1.In one particular embodiment, the bacterial strain of yeast saccharomyces cerevisiae is PE-2.In another particular, the bacterial strain of yeast saccharomyces cerevisiae is CAT-1.In another particular, the bacterial strain of yeast saccharomyces cerevisiae is BG-1.

In some embodiments, cell is monoploid microorganism cells.In other embodiments, cell is diploid microorganism cells.In some embodiments, cell is heterozygosis.In other embodiments, cell except its Alleles of mating type be isozygoty (if namely cell can form spore, so gained four monoploid microorganism cellss can be that heredity is identical except its Alleles of mating type, and described Alleles of mating type can be mating type a and can be mating type α in another two haploid cells in two haploid cells).

In some embodiments, cell is the cell being applicable to industrial fermentation (biological example ethanol fermentation).In specific embodiments, by cell conditioned be generally acknowledged stressed condition high solvent concentration at industrial fermentation environment, high temperature, the substrate utilization expanded, nutrition limits, osmotic pressure (osmotic stress due), acidity should be had, sulphite and bacterial contamination, or the lower existence of its combination.

Hereafter provide exemplary non-alienation compound founder cell, such as restructuring generates isoprenoid, polyketone, and the cell of lipid acid, and for generating the method for this type of cell.

5. the generation of isoprenoid

In some embodiments, non-alienation compound is isoprenoid.Isoprenoid is derived from can by the biosynthetic isopentenylpyrophosphate of enzyme (IPP) of mevalonic acid dependency (" MEV ") approach or DX 5-bisphosphate (" DXP ") approach.

5.1 MEV approach

In some embodiments of the method provided in this article, genetically modified microbe comprises one or more heterologous nucleotide sequence of one or more enzymes of coding MEV approach, and one or more isoprenoid compounds realizing raising compared with the parental cell of non-genetic modification generate.

In some embodiments, isoprenoid founder cell comprises coding energy condensation two molecule acetyl-coenzyme A to form the heterologous nucleotide sequence of the enzyme (such as acetyl-CoA thiolase) of acetoacetyl-CoA.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (NC_000913 region: 2324131.2325315; Intestinal bacteria), (D49362; Paracoccus denitrificans (Paracoccusdenitrificans)), and (L20428; Yeast saccharomyces cerevisiae).

In some embodiments, isoprenoid founder cell comprises coding energy condensation acetoacetyl-CoA with another molecule acetyl-CoA to form the heterologous nucleotide sequence of the enzyme (such as HMG-CoA synthase) of 3-hydroxy-3-methyl glutaryl base-CoA (HMG-CoA).The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (NC_001145. complement 19061.20536; Yeast saccharomyces cerevisiae), (X96617; Yeast saccharomyces cerevisiae), (X83882; Arabidopis thaliana (Arabidopsis thaliana)), (AB037907; Kitasatospora griseola), (BT007302; People), and (NC_002758, locus label SAV2546, GeneID 1122571; Streptococcus aureus (Staphylococcus aureus)).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that HMG-CoA can be transformed into the enzyme (such as HMG-CoA reductase) of mevalonic acid by coding.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (NM_206548; Drosophila melanogaster (Drosophila melanogaster)), (NC_002758, locus label SAV2545, GeneID1122570; Streptococcus aureus), (NM_204485; Jungle fowl (Gallus gallus)), (AB015627; Streptomycete KO 3988), (AF542543; Nicotiana attenuata), (AB037907; Kitasatospora griseola), (AX128213, provides the sequence of coding brachymemma HMGR; Yeast saccharomyces cerevisiae), and (NC_001145: complement 115734.118898; Yeast saccharomyces cerevisiae).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that mevalonic acid can be transformed into the enzyme (such as Mevalonic kinase) of mevalonic acid 5-phosphoric acid by coding.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (L77688; Arabidopis thaliana), and (X55875; Yeast saccharomyces cerevisiae).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that mevalonic acid 5-phosphoric acid can be transformed into the enzyme (such as Phosphomevalonic kinase) of mevalonic acid 5-tetra-sodium by coding.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (AF429385; Para rubber tree (Hevea brasiliensis)), (NM_006556; People) and (NC_001145. complement 712315.713670; Yeast saccharomyces cerevisiae).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that mevalonic acid 5-tetra-sodium can be transformed into the enzyme (such as mevalonate pyrophosphate decarboxylase) of IPP by coding.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (X97557; Yeast saccharomyces cerevisiae), (AF290095; Faecium (Enterococcus faecium)) and (U49260; People).

In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence exceeding a kind of enzyme of coding MEV approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of two kinds of enzymes of coding MEV approach.In some embodiments, isoprenoid founder cell comprises coding and HMG-CoA can be transformed into the enzyme of mevalonic acid and mevalonic acid can be transformed into one or more heterologous nucleotide sequence of the enzyme of mevalonic acid 5-phosphoric acid.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of three kinds of enzymes of coding MEV approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of four kinds of enzymes of coding MEV approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of five kinds of enzymes of coding MEV approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of six kinds of enzymes of coding MEV approach.

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that the IPP generated through MEV approach can be transformed into the enzyme of its isomer dimethylallylpyrophosphate (" DMAPP ") by coding further.Via the effect condensation of other enzyme various and DMAPP can be modified to form simple and more complicated isoprenoid (Fig. 2).

5.2 DXP approach

In some embodiments of the method provided in this article, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of one or more enzymes of encoding D XP approach, and one or more isoprenoid compounds realizing raising compared with the parental cell of non-genetic modification generate.

In some embodiments, isoprenoid founder cell comprises coding energy condensation two molecule acetyl-coenzyme A to form the heterologous nucleotide sequence of the enzyme (such as acetyl-CoA thiolase) of acetoacetyl-CoA.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (NC_000913REGION:2324131.2325315; Intestinal bacteria), (D49362; Paracoccus denitrificans), and (L20428; Yeast saccharomyces cerevisiae).

In some embodiments, isoprenoid founder cell comprise coding can condensation pyruvic acid and D-glyceraldehyde 3 phosphate to form the heterologous nucleotide sequence of the enzyme (such as DX-5-phosphate synthase) of DX-5-phosphoric acid.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (AF035440; Intestinal bacteria), (NC_002947, locus label PP0527; Pseudomonas putida (Pseudomonas putida) KT2440), (CP000026, locus label SPA2301; Salmonella enterica Paratyphi, see ATCC 9150), (NC_007493, locus label RSP_0254; Hydrogenlike silicon ion (Rhodobacter sphaeroides) 2.4.1), (NC_005296, locus label RPA0952; Rhodopseudomonas palustris (Rhodopseudomonaspalustris) CGA009), (NC_004556, locus label PD1293; Xylella fastidiosaTemecula1) and (NC_003076, locus label A T5G11380; Arabidopis thaliana).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that DX-5-phosphoric acid can be transformed into the enzyme (such as DX-5-phosphoric acid reduction isomerase) of 2C-methyl D-erythritol-4-phosphoric acid by coding.The illustrative example of nucleotide sequence includes but not limited to: (AB013300; Intestinal bacteria), (AF148852; Arabidopis thaliana), (NC_002947, locus label PP1597; Pseudomonas putida KT2440), (AL939124, locus label SCO5694; Streptomyces coelicolor A3 (2)), (NC_007493, locus label RSP_2709; Hydrogenlike silicon ion 2.4.1), and (NC_007492, locus label Pfl_1107; Pseudomonas fluorescens (Pseudomonasfluorescens) PfO-1).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that 2C-methyl D-erythritol-4-phosphoric acid can be transformed into the enzyme (such as 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol synthase) of 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol by coding.The illustrative example of nucleotide sequence includes but not limited to: (AF230736; Intestinal bacteria), (NC_007493, locus label RSP_2835; Hydrogenlike silicon ion 2.4.1), (NC_003071, locus label A T2G02500; Arabidopis thaliana), and (NC_002947, locus label PP1614; Pseudomonas putida KT2440).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol can be transformed into the enzyme (such as 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol kinase) of 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol-2-phosphoric acid by coding.The illustrative example of nucleotide sequence includes but not limited to: (AF216300; Intestinal bacteria) and (NC_007493, locus label RSP_1779; Hydrogenlike silicon ion 2.4.1).

In some embodiments, isoprenoid founder cell comprises coding can be transformed into 2C-methyl D-erythritol 2 by 4-cytidine diphosphate (CDP)-2C-methyl D-erythritol-2-phosphoric acid, the heterologous nucleotide sequence of the enzyme (such as 2C-methyl D-erythritol 2,4-ring diphosphate synthase) of 4-ring bisphosphate.The illustrative example of nucleotide sequence includes but not limited to: (AF230738; Intestinal bacteria), (NC_007493, locus label RSP_6071; Hydrogenlike silicon ion 2.4.1), and (NC_002947, locus label PP1618; Pseudomonas putida KT2440).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that 2C-methyl D-erythritol 2,4-ring bisphosphate can be transformed into the enzyme (such as 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase) of 1-hydroxy-2-methyl-2-(E)-butenyl-4-bisphosphate by coding.The illustrative example of nucleotide sequence includes but not limited to: (AY033515; Intestinal bacteria), (NC_002947, locus label PP0853; Pseudomonas putida KT2440) and (NC_007493, locus label RSP_2982; Hydrogenlike silicon ion 2.4.1).

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that 1-hydroxy-2-methyl-2-(E)-butenyl-4-bisphosphate can be transformed into the enzyme (such as isopentyl/dimethylallyl diphosphate synthase) of IPP or its isomer DMAPP by coding.The illustrative example of nucleotide sequence includes but not limited to: (AY062212; Intestinal bacteria) and (NC_002947, locus label PP0606; Pseudomonas putida KT2440).

In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence exceeding a kind of enzyme of encoding D XP approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of two kinds of enzymes of encoding D XP approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of three kinds of enzymes of encoding D XP approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of four kinds of enzymes of encoding D XP approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of five kinds of enzymes of encoding D XP approach.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of six kinds of enzymes of encoding D XP approach.In some embodiments, isoprenoid founder cell comprise encoding D XP approach one or more heterologous nucleotide sequence of five kinds of enzymes.In some embodiments, isoprenoid founder cell comprises one or more heterologous nucleotide sequence of seven kinds of enzymes of encoding D XP approach.

In some embodiments, " crosstalk " (the cross talk) (or the interference) metabolic process of host cell self and those involved between process that IPP generates minimizes or eliminates completely.Such as, when host microorganism body only depends on DXP approach to synthesize IPP by crosstalk minimization or eliminate completely, and MEV approach is introduced to provide other IPP.This kind of host organisms can not be made to be provided as the expression of change MEV path enzyme or to process the intermediate relevant with MEV approach.Only to depend on or organism that mastery depends on DXP approach comprises such as intestinal bacteria.

In some embodiments, host cell, via MEV approach, generates IPP individually or with DXP approach in combination.In other embodiments, make the functional anergy of DXP approach of host, the MEV approach that host cell is only introduced via allos generates IPP.By making genetic expression anergy or the functionally inactive of one or more DXP path enzyme, the functional anergy of DXP approach can be made.

In some embodiments, be C by the isoprenoid of Hemapoiesis ₅isoprenoid.These compounds derive from an isoprene unit and are also called hemiterpene.An illustrative example of hemiterpene is isoprene.In other embodiments, isoprenoid is C ₁₀isoprenoid.These compounds derive from two isoprene units and are also called monoterpene.The illustrative example of monoterpene is limonene, geraniol (citranellol), lemonol, menthol (menthol), perillalcohol (perillyl alcohol), phantol, thujone (thujone), and myrcene.In other embodiments, isoprenoid is C ₁₅isoprenoid.These compounds derive from three isoprene units and are also called sesquiterpene.The illustrative example of sesquiterpene is periplanone (periplanone) B, bilobalide (gingkolide) B, false indigo diene, Artemisinin, Qing Hau acid, valencene, nootkatone, epi-cedrol, epi-aristolochene, farnesol, gossypol (gossypol), sanonin, periplanone, forskolin (forskolin), and Patchoulicalcohol (it is also referred to as Patehouli aleohal (patchouli alcohol)).In other embodiments, isoprenoid is C ₂₀isoprenoid.These compounds derive from four isoprene units and are also called diterpene.The illustrative example of diterpene is castor-oil plant alkene (casbene), eleutherobin, Pa Litasai (paclitaxel), prostratin, pseudopterosin (pseudopterosin), and Japanese yew diene (taxadiene).Also having in other example, isoprenoid is C ₂₀₊isoprenoid.These compounds are from deriving more than four isoprene units and comprising: triterpene is (from the C that 6 isoprene units are derivative ₃₀isoprenoid compounds) such as arbrusideE, bruceantin (bruceantin), testosterone, progesterone, cortisone (cortisone), digoxigenin (digitoxin), and MF59 (squalene); Tetraterpene is (from the C that 8 isoprenoids are derivative ₄₀isoprenoid compounds) such as β-carotene; With polyterpene (certainly derivative more than 8 isoprene units C ₄₀₊isoprenoid compounds) such as polyisoprene.In some embodiments, isoprenoid is selected from lower group: rosin diene, false indigo diene, carene, α-farnesene, β-farnesene, farnesol, lemonol, yak base lemonol, isoprene, phantol, limonene, myrcene, nerolidol, ocimene, Patchoulicalcohol, beta-pinene, sabinene, γ-terpinene, terpinolene and valencene.Isoprenoid compounds also includes but not limited to carotenoid (such as Lyeopene, α-and β-carotene, α-and beta-cryptoxanthin (cryptoxanthin), bixin (bixin), zeaxanthin (zeaxanthin), trans-Astaxanthin (astaxanthin), with xenthophylls (lutein)), sterid, with the compound be made up of the isoprenoid modified through other chemical group, such as mix terpene-alkaloid, and coenzyme q-10.

In some embodiments, isoprenoid founder cell comprises the heterologous nucleotide sequence that the IPP generated through MEV approach can be transformed into the enzyme (such as IPP isomerase) of DMAPP by coding further.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (NC_000913,3031087.3031635; Intestinal bacteria) and (AF082326; Haematocoocus Pluvialls (Haematococcuspluvialis)).

In some embodiments, isoprenoid founder cell comprise further coding can condensation IPP and/or DMAPP molecule to form the heterologous nucleotide sequence containing the polyisoprene synthase of the polyisoprene compound more than five carbon.

In some embodiments, isoprenoid founder cell comprises coding energy condensation a part IPP and a part DMAPP to form the heterologous nucleotide sequence of the enzyme (such as GPP synthase) of a part yak base tetra-sodium (" GPP ").The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (AF513111; Abies grandis), (AF513112; Abies grandis), (AF513113; Abiesgrandis), (AY534686; Common Snapdragon (Antirrhinum majus)), (AY534687; Common Snapdragon), (Y17376; Arabidopis thaliana), (AE016877, locus AP11092; Bacillus cereus; ATCC14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ips pini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha arvensis L. syn.M.haplocalyxBrig (Mentha x piperita)), (AF182827; Mentha arvensis L. syn.M.haplocalyxBrig), (MPI249453; Mentha arvensis L. syn.M.haplocalyxBrig), (PZE431697, locus CAD24425; Paracoccus zeaxanthinifaciens), (AY866498; Picrorhiza kurrooa), (AY351862; Vitis vinifera), and (AF203881, locus AAF12843; Zymomonas mobilis).

In some embodiments, isoprenoid founder cell comprises coding energy condensation two molecule I PP and a part DMAPP, or adds a part IPP to a part GPP to form the heterologous nucleotide sequence of the enzyme (such as FPP synthase) of a part farnesyl pyrophosphate (" FPP ").The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (ATU80605; Arabidopis thaliana), (ATHFPS2R; Arabidopis thaliana), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; E. coli k-12), (AE009951, locus AAL95523; Fusobacterium nucleatumsubsp.nucleatum ATCC 25586), (GFFPPSGEN; Gibberella fujikuroi), (CP000009, locus AAW60034; Gluconobacter oxydans 621H), (AF019892; Helianthus annuus), (HUMFAPS; People), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPS1; Partheniumargentatum), (PAFPS2; Parthenium argentatum), (RATFAPS; Rattusnorvegicus), (YSCFPP; Yeast saccharomyces cerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, locus AAT87386; Streptococcus pyogenes), (CP000017, locus AAZ51849; Streptococcus pyogenes), (NC_008022, locus YP_598856; Streptococcuspyogenes MGAS10270), (NC_008023, locus YP_600845; Streptococcuspyogenes MGAS2096), (NC_008024, locus YP_602832; Streptococcuspyogenes MGAS10750), (MZEFPS; Zea mays (Zea mays)), (AE000657, locus AAC06913; Aquifex aeolicus VF5), (NM_202836; Arabidopis thaliana), (D84432, locus BAA12575; Subtilis (Bacillus subtilis)), (U12678, locus AAC28894; Bradyrhizobium japonicum USDA 110), (BACFDPS; Geobacillusstearothermophilus), (NC_002940, locus NP_873754; Haemophilus ducreyi35000HP), (L42023, locus AAC23087; Haemophilus influenzae Rd KW20), (J05262; People), (YP_395294; Lactobacillus sakei subsp.sakei 23K), (NC_005823, locus YP_000273; Leptospira interrogans serovar Copenhagenistr.Fiocruz L1-130), (AB003187; Micrococcus luteus), (NC_002946, locus YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, locus AAB91752; Rhizobium sp.NGR234), (J05091; Yeast saccharomyces cerevisiae), (CP000031, locus AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, locus AAK99890; Streptococcuspneumoniae R6), and (NC_004556, locus NP 779706; Xylella fastidiosaTemecula1).

In some embodiments, isoprenoid founder cell comprises coding further and can combine IPP and DMAPP or IPP and FPP to form the heterologous nucleotide sequence of the enzyme of yak base yak base tetra-sodium (" GGPP ").The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: (ATHGERPYRS; Arabidopis thaliana), (BT005328; Arabidopis thaliana), (NM_119845; Arabidopis thaliana), (NZ_AAJM01000380, locus ZP_00743052; Bacillus thuringiensis serovarisraelensis, ATCC 35646 sq1563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, locus ZP_00144509; Fusobacterium nucleatum subsp.vincentii, ATCC 49256), (GFGGPPSGN; Gibberella fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Para rubber tree), (AB017971; People), (MCI276129; Mucor circinelloides f.lusitanicus), (AB016044; Mus musculus), (AABX01000298, locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, locus ZP_00943566; Ralstoniasolanacearum UW551), (AB118238; Rattus norvegicus), (SCU31632; Yeast saccharomyces cerevisiae), (AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, locus YP_461832; Syntrophus aciditrophicus SB), (NC_006840, locus YP_204095; Vibriofischeri ES114), (NM_112315; Arabidopis thaliana), (ERWCRTE; Pantoea agglomerans), (D90087, locus BAA14124; Pantoea ananatis), (X52291, locus CAA36538; Rhodobacter capsulatus), (AF195122, locus AAF24294; Hydrogenlike silicon ion) and (NC_004350, locus NP_721015; Streptococcus mutans UA159).

In some embodiments, isoprenoid founder cell comprises coding further and can modify polyisoprene to form hemiterpene, monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, polyterpene, sterid, carotenoid, or the heterologous nucleotide sequence of the enzyme of modified isoprenoid compounds.

In some embodiments, heterologous nucleotide acid encoding carene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (AF461460, REGION 43.1926; Picea excelsa (Picea abies)) and (AF527416, REGION:78.1871; Salvia stenophylla).

In some embodiments, heterologous nucleotide acid encoding lemonol synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (AJ457070; Cinnamomum tenuipilum), (AY362553; Ocimum basilicum), (DQ234300; Perilla frutescens bacterial strain 1864), (DQ234299; Perilla citriodora bacterial strain 1861), (DQ234298; Perilla citriodora bacterial strain 4935) and (DQ088667; Perilla citriodora).

In some embodiments, heterologous nucleotide acid encoding linalool synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (AF497485; Arabidopis thaliana), (AC002294, locus AAB71482; Arabidopis thaliana), (AY059757; Arabidopis thaliana), (NM_104793; Arabidopis thaliana), (AF154124; Artemisia annua), (AF067603; Clarkia breweri), (AF067602; Clarkia concinna), (AF067601; Clarkia breweri), (U58314; Clarkia breweri), (AY840091; Lycopersicon esculentum), (DQ263741; Lavandula angustifolia), (AY083653; Mentha citrate), (AY693647; Ocimum basilicum), (XM_463918; Oryza sativa), (AP004078, locus BAD07605; Oryza sativa), (XM_463918, locus XP_463918; Oryza sativa), (AY917193; Perilla citriodora), (AF271259; Perilla frutescens), (AY473623; Picea excelsa), (DQ195274; And (AF444798 Piceasitchensis); Perilla frutescens var.crispa cultivar No.79).

In some embodiments, heterologous nucleotide acid encoding limonene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (+)-limonene synthase (AF514287, REGION:47.1867; Citrus limon) and (AY055214, REGION:48.1889; Agastache rugosa) and (-)-limonene synthase (DQ195275, REGION:1.1905; Picea sitchensis), (AF006193, REGION:73.1986; Abies grandis), and (MHC4SLSP, REGION:29.1828; Menthaspicata).

In some embodiments, heterologous nucleotide acid encoding myrcene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (U87908; Abies grandis), (AY195609; Common Snapdragon), (AY195608; Common Snapdragon), (NM_127982; Arabidopis thaliana TPS10), (NM_113485; Arabidopis thaliana ATTPS-CIN), (NM_113483; Arabidopis thaliana ATTPS-CIN), (AF271259; Perillafrutescens), (AY473626; Picea excelsa), (AF369919; Picea excelsa), and (AJ304839; Quercus ilex).

In some embodiments, heterologous nucleotide acid encoding ocimene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (AY195607; Common Snapdragon), (AY195609; Common Snapdragon), (AY195608; Common Snapdragon), (AK221024; Arabidopis thaliana), (NM_113485; Arabidopis thaliana ATTPS-CIN), (NM_113483; Arabidopis thaliana ATTPS-CIN), (NM_117775; Arabidopis thaliana ATTPS03), (NM_001036574; Arabidopis thaliana ATTPS03), (NM_127982; Arabidopis thaliana TPS10), (AB110642; Citrus unshiu CitMTSL4), and (AY575970; Lotuscorniculatus var.japonicus).

In some embodiments, HETEROLOGOUS NUCLEOTIDE coding α-pinene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (+) α-pinene synthase (AF543530, REGION:1.1887; Pinustaeda), (-) α-pinene synthase (AF543527, REGION:32.1921; Pinus taeda), and (+)/(-) α-pinene synthase (AGU87909, REGION:6111892; Abies grandis).

In some embodiments, heterologous nucleotide acid encoding beta-pinene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (-) beta-pinene synthase (AF276072, REGION:1.1749; Artemisia annua) and (AF514288, REGION:26.1834; Citrus limon).

In some embodiments, heterologous nucleotide acid encoding sabinene synthase.An illustrative example of appropriate nucleotide sequence includes but not limited to: AF051901, REGION:26.1798, from Salviaofficinalis.

In some embodiments, heterologous nucleotide acid encoding γ-terpinene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (AF514286, REGION:30.1832, from Citrus limon) and (AB110640, REGION 1.1803, from Citrus unshiu).

In some embodiments, heterologous nucleotide acid encoding terpinolene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (AY693650, from Oscimum basilicum) and (AY906866, REGION:10.1887, from Pseudotsuga menziesii).

In some embodiments, HETEROLOGOUS NUCLEOTIDE encoding amorpha diene synthase.An illustrative example of appropriate nucleotide sequence is the SEQ ID NO.37 of U.S. Patent Publication text No.2004/0005678.

In some embodiments, HETEROLOGOUS NUCLEOTIDE coding for alpha-farnesene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to the DQ309034 (pears from Pyrus communis cultivar d'Anjou; Gene name AFS1) and from the AY182241 (apple of Malus domestica; Gene A FS1).Pechouus et al.,Planta219(1):84-94(2004)。

In some embodiments, heterologous nucleotide acid encoding β-farnesene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to the GenBank accession number AF024615 (peppermint from Mentha arvensis L. syn.M.haplocalyxBrig; Gene Tspa11), and from the AY835398 of Artemisia annua.Picaud et al.,Phytochemistry66(9):961-967(2005)。

In some embodiments, HETEROLOGOUS NUCLEOTIDE coding farnesol synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to from zeistic GenBank accession number AF529266 and the YDR481C (gene Pho8) from yeast saccharomyces cerevisiae.Song,L.,Applied Biochemistry andBiotechnology128:149-158(2006)。

In some embodiments, heterologous nucleotide acid encoding nerolidol synthase.A kind of illustrative example of appropriate nucleotide sequence includes but not limited to from zeistic AF529266 (corn; Gene tps1).

In some embodiments, heterologous nucleotide acid encoding Patchoulicalcohol synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: the AY508730 region from Pogostemon cablin: 1.1659.

In some embodiments, heterologous nucleotide acid encoding nootkatone synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: the AF441124 region from Citrus sinensis: 1.1647 and AY917195 region from Perilla frutescens: 1.1653.

In some embodiments, heterologous nucleotide acid encoding rosin diene synthase.The illustrative example of appropriate nucleotide sequence includes but not limited to: (U50768; Abies grandis) and (AY473621; Picea excelsa).

6. the generation of polyketone

In some embodiments, non-alienation compound is polyketone.Polyketone is by by the sequential Reactive Synthesis of one group of enzymic activity catalysis being called polyketide synthase (PKS) (they coordinate the large-scale multienzyme albumen composition of avtive spot containing a group).Polyketone biosynthesizing progressively moves ahead, start from simple 2-, 3-, 4-carbon builds block, such as acetyl-CoA, propionyl-CoA, butyryl-CoA and activated derivatives thereof, malonyl--, methylmalonyl-and ethyl malonyl--CoA, mainly via the decarboxylation condensation of malonyl--CoA derived units through Claisen condensation reaction.PKS gene forms into an operon usually in bacterium, in eukaryote, be organized into gene cluster.Characterized the polyketide synthase of three types: I type polyketide synthase is large-scale, high modularization albumen, be subdivided into two classifications: 1) repeated PKS, they re-use structural domain in a looping fashion; With 2) modularization PKS, they contain a series of module of separating and do not have repeating structure territory.II type polyketide synthase is the aggregation of single functional protein, and type III polyketide synthase does not use acyl carrier protein territory.

Synthesize (wherein as described below with fatty acid biological, each continuous chain extends step and continues with the ketone body powder of fixed series, dehydration and alkene acyl, reduction) different, polyketone each chain extension intermediate biosynthetic experience is all, experience, or do not experience modified with functional group, produce a lot of chemically various product.The complicacy of degree is derived from and uses different start element and chain extension unit and generate new steric isomer in addition.

Order following (N end is to the order of C end) by the complete polyketone synthesis that polyketide synthase instructs: initial or initial carbon is built block and be loaded on acyl carrier protein, extends the macrolide chain extension in module catalytic growth and stops the macrolide release that module catalyzes and synthesizes.Activated component territory or the enzyme that separates is functional comprises acyltransferase (it is for loading initiator, continuation and middle acyl unit) in this biosynthesizing; Acyl carrier protein (it keeps the macrolide in growth with mercaptan ester-formin); β-one-acyl group synthase (its catalysis chain extension); Beta-Keto-reductase (it is responsible for first being reduced into alcohol functionalities); Dehydratase (it eliminates water to provide unsaturated thioesters); Enoyl reductase (its catalysis is finally reduced to completely saturated); With thioesterase (release of its catalysis macrolide and cyclisation).

In some embodiments, useful to method disclosed herein genetically modified microbe comprises the heterologous nucleotide sequence of the enzyme (such as acyltransferase) of at least one item and acyl carrier protein in coding energy condensation acetyl-CoA and malonyl--CoA.

In some embodiments, genetically modified microbe disclosed herein comprise coding can condensation the first reactant of being selected from acetyl-CoA and malonyl--CoA be selected from second reactant of malonyl--CoA or methylmalonyl-CoA to form the heterologous nucleotide sequence of the enzyme (such as β-one-acyl group synthase) of polyketone product.

In some embodiments, polyketone founder cell comprises the heterologous nucleotide sequence that the beta-keto chemical group on polyketide can be reduced into the enzyme (such as beta-Keto-reductase) of beta-hydroxy group by coding.

In some embodiments, genetically modified microbe disclosed herein comprises the heterologous nucleotide sequence of enzyme (such as dehydratase) that coding can make the alkane chemical group in polyketide dewater to generate alpha-beta-unsaturated olefin.

In some embodiments, polyketone founder cell comprises the heterologous nucleotide sequence that the alpha-beta-reduction in polyketide can be become the enzyme (such as enoyl reductase) of saturated alkane by coding.

In some embodiments, polyketone founder cell comprise coding can from the heterologous nucleotide sequence of the enzyme of acyl carrier protein hydrolyzed polyketone compound (such as thioesterase).

In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of KS catalytic domain.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of AT catalytic domain.In some embodiments, polyketone founder cell comprise encoded packets containing AT catalytic domain enzyme exceed a kind of heterologous nucleotide sequence.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of CLF catalytic domain.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of ACP activity.In some embodiments, polyketone founder cell comprise encoded packets containing ACP activity enzyme exceed a kind of heterologous nucleotide sequence.

In one particular embodiment, polyketone founder cell comprises bottom line aromatic series PKS system, and such as respectively encoded packets, containing the enzyme of KS catalytic domain, comprises the enzyme of AT catalytic domain, comprises the enzyme of CLF catalytic domain, and comprises the heterologous nucleotide sequence of enzyme of ACP activity.In one particular embodiment, polyketone founder cell comprises bottom line modularization PKS system, and such as respectively encoded packets, containing the enzyme of KS catalytic domain, comprises the enzyme of AT catalytic domain, and comprises the heterologous nucleotide sequence of enzyme of ACP activity.Also having in another particular, polyketone founder cell comprises the modularization aromatic series PKS system for polyketone synthesis again, such as encoded packets contains the enzyme of KS catalytic domain respectively, comprise one or more enzymes of AT catalytic domain, and comprise the heterologous nucleotide sequence of one or more enzymes of ACP activity.

In some embodiments, polyketone founder cell comprises bottom line PKS system, such as bottom line aromatic series PKS system or bottom line modularization PKS system, comprises further and end product can be facilitated to gather other catalytic activity ketogenetic.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of cyclase (CYC) catalytic domain (it is backbone cyclized that it promotes newborn polyketone).In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of ketoreductase (KR) catalytic domain.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of aromatase enzyme (ARO) catalytic domain.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of enoyl reductase (ER) catalytic domain.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of thioesterase (TE) catalytic domain.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of full ACP synthase activity (it realizes the pantetheine (pantetheinylation) of ACP) further.

In some embodiments, polyketone founder cell comprises one or more heterologous nucleotide sequence of giving the rear polyketone modification activities of synthesis further.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence of enzyme containing glycosylation enzymic activity (its synthesis realizing polyketone is modified afterwards) further, such as, have in the polyketone situation of Antibiotic activity wanting.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of hydroxylase activity further.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of cyclooxigenase activity further.In some embodiments, polyketone founder cell comprises encoded packets one or more heterologous nucleotide sequence containing the enzyme of methylase activity further.

In some embodiments, polyketone founder cell comprises one or more heterologous nucleotide sequence of encoding biosynthetic enzymes further, include but not limited at least one polyketone route of synthesis enzyme, and acetyl-CoA compound can be modified to form polyketone product (such as macrolide, biocide, anti-mycotic agent, T suppression cell compound, Anticholesterolemic compound, anti-parasitic compound, suppress coccidia compound, animal growth promoter or sterilant) enzyme.In some embodiments, non-alienation compound is polyenoid.In some embodiments, non-alienation compound is cyclic lactone.In some embodiments, non-alienation compound comprises 14,15, or 16 yuan of lactonic rings.In some embodiments, non-alienation compound is be selected from the polyketone of lower group: polyketone macrolide, biocide, anti-mycotic agent, T suppression cell compound, Anticholesterolemic compound, anti-parasitic compound, suppresses coccidia compound, animal growth promoter and sterilant.

In some embodiments, polyketone founder cell comprises heterologous nucleotide sequence, such as coding can generate and is selected from but is not limited to the PKS enzyme of the polyketone of following polyketone and the sequence of polyketone modifying enzyme: avermectin (Avermectin) is (see such as U.S. Patent No. 5,252,474; U.S. Patent No. 4,703,009; The open text No.118 in Europe, 367; MacNeil et al., 1993, " Industrial Microorganisms:Basic and Applied Molecular Genetics "; Baltz, Hegeman, & Skatrud, eds. (ASM), pp.245-256, " A Comparison of the Genes Encoding the PolyketideSynthases for Avermectin; Erythromycin, and Nemadectin "; MacNeil et al., 1992, Gene 115:119-125; And Ikeda and Omura, 1997, Chem.Res.97:2599-2609); Candicidin (Candicidin) (FR008) (see such as Hu et al., 1994, Mol.Microbiol.14:163-172); Magnamycin A (Carbomycin), curamycin (Curamycin) is (see such as Bergh etal., Biotechnol Appl Biochem.1992Feb; 15 (1): 80-9); Daunomycin (Daunorubicin) is (see such as J Bacteriol.1994Oct; 176 (20): 6270-80); Esperamicin (Epothilone) is (see the open text No.99/66028 of such as PCT; And the open text No.00/031247 of PCT); Erythromycin (Erythromycin) is (see the open text No.93/13663 of such as PCT; U.S. Patent No. 6,004,787; U.S. Patent No. 5,824,513; Donadio et al., 1991, Science 252:675-9; And Cortes et al., Nov.8,1990, Nature 348:176-8); FK-506 (see such as Motamediet al., 1998; Eur.J Biochem.256:528-534; And Motamedi et al., 1997, Eur.JBiochem.244:74-80); FK-520 is (see the open text No.00/020601 of such as PCT; And Nielsen et al., 1991, Biochem.30:5789-96); Spirofulvin (Griseusin) is (see such as Yuet al., J Bacteriol.1994 May; 176 (9): 2627-34); Lovastatin (Lovastatin) (see such as U.S. Patent No. 5,744,350); Frenolycin is (see such as Khosla et al., Bacteriol.1993Apr; 175 (8): 2197-204; And Bibb et al., Gene 1994 May 3; 142 (1): 31-9); (see such as Sherman et al., EMBO is Sep J.1989 for litmomycin (Granaticin); 8 (9): 2717-25; And Bechtold et al., Mol Gen Genet.1995 Sep 20; 248 (5): 610-20); Medermycin (Medermycin) is (see such as Ichinose et al., Microbiology 2003 Jul; 149 (Pt7): 1633-45); Monensin (Monensin) is (see such as Arrowsmith et al., Mol Gen Genet.1992 Aug; 234 (2): 254-64); Nonactin (Nonactin) is (see such as FEMS Microbiol Lett.2000 Feb 1; 183 (1): 171-5); Rosanomycin (Nanaomycin) is (see such as Kitao et al., JAntibiot (Tokyo) .1980 Jul; 33 (7): 711-6); Nemadectin (Nemadectin) (see such as MacNeil et al., 1993, supra); Niddamycin (Niddamycin) is (see the open text No.98/51695 of such as PCT; And Kakavas et al., 1997, J.Bacteriol.179:7515-7522); Romicil (Oleandomycin) is (see such as Swan et al., 1994, Mol.Gen.Genet.242:358-362; The open text No.00/026349 of PCT; Olano et al., 1998, Mol.Gen.Genet.259 (3): 299-308; And the open text No.WO 99/05283 of PCT patent application); Terramycin (Oxytetracycline) is (see such as Kim et al., Gene.1994 Apr 8; 141 (1): 141-2); Pikromycin (Picromycin) is (see the open text No.99/61599 of such as PCT; The open text No.00/00620 of PCT; Xue et al., 1998, Chemistry & Biology 5 (11): 661-667; Xue et al., October 1998, Proc.Natl.Acad.Sci.USA 95:12111 12116); Platenolide (see the open text No.791 in such as Europe, 656; And U.S. Patent No. 5,945,320); Rapamycin (Rapamycin) is (see such as Schwecke et al., August 1995, Proc.Natl.Acad.Sci.USA 92:7839-7843; And Aparicio et al., 1996, Gene 169:9-16); Rifomycin (Rifamycin) is (see the open text No.WO 98/07868 of such as PCT; And August et al., Feb.13,1998, Chemistry & Biology, 5 (2): 69-79); Sorangium (see such as U.S. Patent No. 6,090,601); Soraphen is (see such as U.S. Patent No. 5,716,849; Schupp et al., 1995, J.Bacteriology 177:3673-3679); Spinocyn (see the open text No.99/46387 of such as PCT); Spiramycin Base (Spiramycin) (see such as U.S. Patent No. 5,098,837); Tetracenomycin (Tetracenomycin) is (see such as Summers et al., J Bacteriol.1992Mar; 174 (6): 1810-20; And Shen et al., J Bacteriol.1992 Jun; 174 (11): 3818-21); Tsiklomitsin (Tetracycline) is (see such as J Am Chem Soc.2009 Dec 9; 131 (48): 17677-89); Tylosin (Tylosin) is (see such as U.S. Patent No. 5,876,991; U.S. Patent No. 5,672,497; U.S. Patent No. 5,149,638; The open text No.791 in Europe, 655; The open text No.238 in Europe, 323; Kuhstoss et al., 1996, Gene 183:231-6; And Merson-Davies and Cundliffe, 1994, Mol.Microbiol.13:349-355); And 6-cresotinic acid is (see such as Richardson etal., Metab Eng.1999 Apr; 1 (2): 180-7; And Shao et al., Biochem Biophys ResCommun.2006 Jun 23; 345 (1): 133-9).

7. the generation of lipid acid

In some embodiments, non-alienation compound is lipid acid.By Fatty acid synthetase catalysis, from acetyl-CoA and malonyl--CoA by a series of decarboxylation Claisen condensation reaction synthetic fatty acid.Similar to polyketide synthase, Fatty acid synthetase is not the enzyme system formed with single enzyme but by 272kDa multifunctional polypeptides, and wherein substrate gives the next one from a functional domain.Two primary categories of Fatty acid synthetase are characterized: I type Fatty acid synthetase is Mammals and fungi (although the structural arrangement of fungi and Mammals synthase has difference) and CMN group bacterium (coryneform bacteria (corynebacteria), mycobacterium (mycobacteria), and Nocardia bacteria (nocardia)) common a kind of multifunctional polypeptides.The II type synthase found in archeobacteria and eubacterium is the series of discrete participating in lipid acid synthesis, single functional enzyme.The mechanism of fatty acid prolonging and shortening is identical in two class synthase, because be responsible for the enzyme domains of these catalyzed event is largely homology between two classes.

After often wheel fatty acid chain in decarboxylation Claisen condensation reaction extends, beta-keto group passes through ketoreductase, dehydratase, and the sequential effect of enoyl reductase is reduced into complete saturated carbon chains.Fatty acid chain in growth moves between these avtive spots being attached to acyl carrier protein, is finally discharged by the effect of thioesterase reaching carbon chain lengths 16 (palmitinic acid (palmitidic acid)) time.

In some embodiments, the genetically modified microbe useful to method disclosed herein comprises the heterologous nucleotide sequence of encoding biosynthetic enzymes, includes but not limited at least one fatty acid synthesis pathway enzyme, with can modify acetyl-CoA compound to form fatty acids products (such as palmitinic acid, palmityl CoA, Zoomeric acid, fragrant juniperic acid, oleic acid, linolic acid, alpha-linolenic acid, arachidonic acid, timnodonic acid, erucic acid, and docosahexenoic acid) enzyme.In some embodiments, non-alienation compound is be selected from the lipid acid of lower group: palmitinic acid, palmityl CoA, Zoomeric acid, fragrant juniperic acid, oleic acid, linolic acid, alpha-linolenic acid, arachidonic acid, timnodonic acid, erucic acid, and docosahexenoic acid.

In some embodiments, lipid acid founder cell comprises the heterologous nucleotide sequence of the enzyme (such as acyltransferase) of at least one item and acyl carrier protein in coding energy covalently bound acetyl-CoA and malonyl--CoA.

In some embodiments, genetically modified microbe disclosed herein comprise coding can condensation acetyl chemical module and malonyl-chemical module (being bonded to acyl carrier protein (ACP) separately) to form the heterologous nucleotide sequence of the enzyme (such as beta-keto acyl base-ACP synthase) of acetoacetyl-ACP.

In some embodiments, lipid acid founder cell comprises the double bond that can reduce in acetoacetyl-ACP with NADPH of encoding to form the heterologous nucleotide sequence of the enzyme (such as beta-keto acyl base-ACP reductase enzyme) of the oh group in D-3-maloyl group hydroxylase-ACP.

In some embodiments, lipid acid founder cell comprises the heterologous nucleotide sequence that coding can make D-3-maloyl group hydroxylase-ACP dewater to produce the enzyme (such as beta-hydroxy acyl-ACP dehydratase) of double bond between the β-forming crotonyl-ACP and γ-carbon.

In some embodiments, lipid acid founder cell comprise coding can reduce crotonyl-ACP to form the heterologous nucleotide sequence of the enzyme (such as alkene acyl-ACP reductase enzyme) of butyryl radicals-ACP with NADPH.

In some embodiments, lipid acid founder cell comprises coding and can be hydrolyzed C from acyl carrier protein ₁₆acyl compounds is to form the heterologous nucleotide sequence of the enzyme (such as thioesterase) of palmitinic acid.

In some embodiments, lipid acid founder cell comprises one or more heterologous nucleotide sequence of encoding acetyl-CoA synthase and/or malonyl--CoA synthase, generates to realize one or more lipid acid of rising compared with the parental cell of non-genetic modification.

Such as; generate to improve acetyl-CoA; can at one or more following gene: pdh of cells; panK, aceEF (the EIp desaturase component of encode pyruvate and odhA mixture and E2p dihydrolipoamide acyltransferase component), fabH; fabD; fabG, acpP, and fabF.The illustrative example of nucleotide sequence of this fermentoid of encoding includes but not limited to: pdh (BAB34380, AAC73227, AAC73226), panK is (also referred to as coaA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), fabF (AAC74179).

In some embodiments, by weakening or knock out the gene that coding relates to the protein of Fatty acid degradation, the fatty acid levels raised can be realized in cell.Such as, technology known in the art can be used to weaken in engineered host cell or knock out fadE, gpsA, idhA, pflb, adhE, pta, poxB, ackA, and/or the expression level of ackB.The illustrative example of the nucleotide sequence of encoding such proteinaceous includes but not limited to: fadE (AAC73325), gspA (AAC76632), IdhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430).When growing in control environment, gained host cell can have the acetyl-CoA generation level of rising.

In some embodiments, lipid acid founder cell comprises the heterologous nucleotide sequence that acetyl-CoA can be transformed into the enzyme (such as many subunits AccABCD albumen) of malonyl--CoA by coding.An illustrative example of the appropriate nucleotide sequence of coding AccABCD includes but not limited to accession number AAC73296, EC6.4.1.2.

In some embodiments, lipid acid founder cell comprises the heterologous nucleotide sequence of encoding lipase.The illustrative example of the appropriate nucleotide sequence of encoding lipase includes but not limited to accession number CAA89087 and CAA98876.

In some embodiments; by suppressing PlsB, (this can cause long acyl-ACP level to improve, and this can suppress early stage step (such as accABCD, fabH in fatty acid biosynthetic pathway; and fabl)), the fatty acid levels raised can be realized in cell.Technology known in the art can be used to weaken in engineered host cell or knock out the expression level of PlsB.An illustrative example of the appropriate nucleotide sequence of coding PlsB includes but not limited to accession number AAC77011.In specific embodiments, plsB D31 IE sudden change can be used for the amount improving available acyl group-CoA in cell.

In some embodiments, by process LAN sfa gene (this can cause fabA to prevent), the monounsaturated fatty acids that can realize raising in cell generates.A kind of illustrative example of the appropriate nucleotide sequence of coding sfa includes but not limited to accession number AAN79592.

In some embodiments, controlled the expression of the enzyme (such as thioesterase) of fatty acid substrate chain length by regulation and control, the fatty acid levels raised can be realized in cell.In some embodiments, lipid acid founder cell has been modified into process LAN tes or fat gene.The illustrative example of suitable tes nucleotide sequence includes but not limited to accession number: (tesA:AAC73596, from intestinal bacteria, can generate C ₁₈: 1 lipid acid) and (tesB:AAC73555, from intestinal bacteria).The illustrative example of suitable fat nucleotide sequence includes but not limited to: (fatB:Q41635 and AAA34215, from Umbellularia california, can generate C ₁₂: 0 lipid acid), (fatB2:Q39513 and AAC49269, from Cupheahookeriana, can generate C ₈: 0 – C ₁₀: 0 lipid acid), (fatB3:AAC49269 and AAC72881, from Cuphea hookeriana, can generate C ₁₄: 0 – C ₁₆: 0 lipid acid), (fatB:Q39473 and AAC49151, from Cinnamonum camphorum, can generate C ₁₄: 0 lipid acid), (fatB [M141T]: CAA85388, from Arabidopis thaliana, can C be generated ₁₆: 1 lipid acid), (fatA:NP 189147 and NP 193041, from Arabidopis thaliana, can generate C ₁₈: 1 lipid acid), (fatA:CAC39106, from Bradvrhiizobium japonicum, preferentially can generate C ₁₈: 1 lipid acid), (fatA:AAC72883, from Cuphea hookeriana, can generate C ₁₈: 1 lipid acid), and (fatA1, AAL79361, from Helianthus annus).

In some embodiments, by using technology known in the art to weaken thioesterase C ₁₈expression or activity, can realize in cell raise C ₁₀fatty acid levels.Coding thioesterase C ₁₈the illustrative example of appropriate nucleotide sequence include but not limited to accession number AAC73596 and P0ADA1.In other embodiments, by using technology known in the art to improve thioesterase C ₁₀expression or activity, can realize in cell raise C ₁₀fatty acid levels.Coding thioesterase C ₁₀an illustrative example of appropriate nucleotide sequence include but not limited to accession number Q39513.

In some embodiments, non-C is generated by using technology known in the art to weaken ₁₄the expression of the endogenous thioesterase of lipid acid or activity, can realize the C raised in cell ₁₄fatty acid levels.In other embodiments, substrate C is used by using technology known in the art to improve ₁₄the expression of the thioesterase of-ACP or activity, can realize the C raised in cell ₁₄fatty acid levels.The illustrative example of appropriate nucleotide sequence of this type of thioesterase of encoding includes but not limited to accession number Q39473.

In some embodiments, non-C is generated by using technology known in the art to weaken ₁₂the expression of the endogenous thioesterase of lipid acid or activity, can realize the C raised in cell ₁₂fatty acid levels.In other embodiments, substrate C is used by using technology known in the art to improve ₁₂the expression of the thioesterase of-ACP or activity, can realize the C raised in cell ₁₂fatty acid levels.The illustrative example of appropriate nucleotide sequence of this type of thioesterase of encoding includes but not limited to accession number Q41635.

Embodiment

Embodiment 1

This embodiment describes a kind of exemplary cell density (OD for measuring yeast cell culture ₆₀₀) method.

8 μ L cell culture sample and 92 μ L Triton OD thinner (20g/L Triton X-114 are combined in transparent 96 orifice plates, 200mL/L PEG 200,200mL/L 100% ethanol, all the other are water), solution is shaken 6 minutes with 1,000RPM, and passes through at M5 spectrophotometer (Molecular Devices, Sunnyvale, CA) in 600nm measure absorbancy measure OD ₆₀₀.

Embodiment 2

This embodiment describes a kind of exemplary method based on Nile red, and its farnesene titre for mensuration yeast cell culture is useful.

98 μ L cell culture sample are transferred to 96 hole black polystyrene flat bottom assay plates, and the Nile red (Invitrogen, Carlsbad, CA) that 2 μ L dissolve with 100 μ g/mL in DMSO is added into each hole.Excite and 550nm emission measurement fluorescence level with 500nm on M5 spectrophotometer immediately.

Embodiment 3

This embodiment describes a kind of exemplary method based on gas chromatography (GC), and its farnesene titre for mensuration yeast cell culture is useful.

Sample is extracted with methyl alcohol-heptane (1:1v/v), and by centrifugal for mixture to remove cell material.Containing 0.001%t-caryophyllene (caryohyllene), (it serves as retention time mark, for monitor during regulation GC stove overview successfully injection and wash-out) n-heptane in dilute a methyl alcohol-heptane extract, then use pulse split injection injection on methyl silicone static phases.The GC that use is furnished with flame ionization detection (FID) is separated farnesene by boiling point.

Embodiment 4

The strain degeneration phenomenon that this embodiment occurs when non-alienation compound is generated as "ON" during proving the construction of zymotechnique and production phase both.

The isodynamic enzyme that comprises melting 1ml phial (comprises MEV path enzyme (Fig. 1): IPP isomerase, FPP synthase, with farnesene synthase) and the frozen cell suspension of the yeast strain of exemplary non-alienation compound (farnesene) can be generated, be transferred to the 250-ml that 50ml BSM 2.0 (containing 2% sucrose and 10mg/LD-calcium pantothenate) is housed and be with baffled flask, and with 34 DEG C in shaking table, 200RPM grows 24 hours.Then whole culture is transferred to the 2.8L Fernbach flask that 850ml BSM 2.0 (containing 2.0% sucrose and 10mg/L D-VB5 calcium) is housed, and with 34 DEG C in shaking table, 250RPM grows 24 hours.Then whole culture is transferred to 2L fermentor tank.The nutrition feed of fermentor tank be comprise 10mg/L D-VB5 calcium do not limit Brazilian sugarcane syrup substratum, deliver with the inceptive impulse being equal to 14g/L/h sugar.Then based on demand (as dissolved oxygen raise shown) the self-control delivery rate of fermentor tank to carbon.With steady temperature 34 DEG C, constant pH 4.5 (controlling by adding sodium hydroxide), and initial oxygen transfer rate 200mmol O ₂the micro-aerobic operation fermentation of/L/h, until dissolved oxygen reaches 0%, then ferments and is reduced to 100mmol O remaining time ₂/ L/h.Every 3 days, the volume of tank is reduced to about 0.9L to prevent from overflowing.Supplement the trace-metal lost in sugarcane syrup feed and VITAMIN at that time.Monitor the growing amount of farnesene and the total reducing sugar of cell consumption every day, for every 72 hours determine the ratio (i.e. products collection efficiency/sugar) of these two numerical value, and draw, as shown in Figure 3.The peak of products collection efficiency from 6 days time of culture drops to 65% of this peak of < to when 21 days.

Embodiment 5

The result that this embodiment provides proves can generate isoprenoid farnesene and be included in micro-aerobic responsiveness promotor (" low O ₂switch ") just regulating under the host cell of MEV approach at high O ₂in condition, (rocker) generates the farnesene of very low amounts, and at low O ₂in condition (low RPM shaking flask), generate substantive be increased to MEV approach constructive expression can not the generation of the switch parent strain level of mating.Result is depicted in Fig. 6.

Farnesene generates yeast strain:

Use a kind of derived from wild type Saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain (CEN.PK2) and " can not the switch " farnesene of expressing the gene (Fig. 1) of mevalonate pathway under GAL promotor controls generate bacterial strain and generate contrast as composition farnesene.This can not switch bacterial strain comprise following chromosomal integration GAL promotor control under the mevalonate pathway gene from yeast saccharomyces cerevisiae: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, Mevalonic kinase, Phosphomevalonic kinase, and mevalonate pyrophosphate decarboxylase; With the farnesene synthase mutant of six copies from sweet wormwood (Artemisinin annua).This can not delete gal80 gene and has the GAL4 under GAL4oc promotor of an additional copy by switch bacterial strain, and wherein the encoding sequence of the GAL4 gene of yeast saccharomyces cerevisiae is at its natural promoter (PGAL4oc; See such as Griggs & Johnston (1991) PNAS 88 (19): 8597-8601) " can operate composition " pattern regulable control under.

Then make the farnesene in this " can not switch " bacterial strain generate " can switch ", namely can prevent under aerobic condition.By replacing the promotor of the GAL4 of multiple copy by DAN1#1 promotor (SEQ ID NO:1), on this composition bacterial strain, build low O ₂switch can switch bacterial strain.By replacing pGAL4oc with pDAN1#1, introduce the pDAN1#1 that first copy drives GAL4.Then natural GAL4 promotor is replaced with pDAN1#1.This bacterial strain is called in figure 6 " low O ₂can switch bacterial strain #1 ".From this bacterial strain, integrate the pDAN1#1 of the 3rd and the 4th copy driving GAL4 at GAS2 locus place simultaneously.This second can be called " low O by switch bacterial strain (it has four GAL4 of copy under DAN1#1 promotor controls) in the figure 7 ₂can switch bacterial strain #2 ".

Non-alienation compound is regulated to generate by changing oxygen condition:

Cultivate under aerobic and micro-aerobic condition respectively and can not generate control strain and low O by switch farnesene ₂can generate to assess the farnesene served as under the fermentation condition of "Off" and "On" state in intention by both switch bacterial strain #1.

For "Off" or " high O ₂condition ", the bacterium colony of picking two kinds of bacterial strains enters the 360uL BSM2.0 in 96 orifice plates, 2% sucrose.Plate is shaken 3 days with high RPM in ATR shaking table.From this preculture plate transfer 6uL culture to production 96 orifice plate that 360uL BSM 2.0,4% sucrose is housed.Plate is shaken 2 days with high RPM in ATR shaking table.Farnesene concentration is measured by GC-FID.

For "ON" or " low O ₂condition ", in 125ml flask, inject 50ml BSM 2.0,4% substratum and inoculate 0.1OD and often plant bacterial strain.50ml is the conventional large culture volume of 125ml flask size, and this contribute to reducing enter flask oxygen transfer rate (OTR) to guarantee that cultivation becomes micro-aerobic.Through probe measurement dissolved oxygen, confirm two kinds of bacterial strains in 24 hours, all reach the dissolved oxygen levels that can't detect.Farnesene concentration is measured by GC-FID after 120 hours.Constantly little 120, the dissolved oxygen of two kinds of cultures is all roughly the desired value of gas-liquid equilibrium, and hint carbon exhausts.

As shown in Figure 6, can not compared with switch bacterial strain with the composition generating high-level farnesene, low O ₂can switch bacterial strain #1 at high O ₂little farnesene is generated under condition.At low O ₂in condition, low O ₂can the farnesene in switch bacterial strain #1 to generate induction higher, and exceeded composition and can not the farnesene of switch bacterial strain generate.These results prove that oxygen operation can be used in as low O ₂can switch, farnesene generates bacterial strain and realizes tight "Off" and "On" state.

Embodiment 6

Compared with result that this embodiment provides proves to produce the generation of bacterial strain with the composition running through the construction phase and generate farnesene, when implementing construction phase (the realizing "Off" state thus) of fermenting under aerobic condition, isoprenoid farnesene can be generated and be included in low O ₂the host cell of the MEV approach under switch is just regulating shows that in long hair ferment runs the farnesene of improvement generates stability.Result is depicted in Fig. 7.

For generating bacterial strain and low O by the switches set farnesene that becomes second nature ₂can both switch bacterial strain #2, melt the frozen cell suspension of 1ml phial, be transferred to the 250-ml that 50ml BSM 3.0 (containing 1.6% sucrose, 0.4% glucose is as carbon source) is housed and be with baffled flask, and with 34 DEG C in shaking table, 250RPM grows 24 hours.Then enter 50ml containing 1.6% sucrose from flask transferase 12 ml, 0.4% glucose is as the BSM 3.0 of carbon source, and with 34 DEG C in shaking table, 250RPM grows 24 hours.Then transferase 12 5ml enters to be equipped with the inoculation bottle of 225ml tank substratum and is transferred to 0.5L fermentor tank.The nutrition feed of fermentor tank is 650g/L sucrose solution, delivers with the inceptive impulse being equal to 10g/L/h sugar.Then based on demand (as dissolved oxygen raise shown) the self-control delivery rate of fermentor tank to carbon.Once dissolved oxygen reaches 0%, with steady temperature 34 DEG C, constant pH 4.5 (controlling by adding sodium hydroxide), and maximum oxygen transfer rate 110mmol O ₂the micro-aerobic operation fermentation of/L/h.Every day, the volume of tank is reduced to about 0.29L to prevent from overflowing.Supplement trace-metal and VITAMIN at that time.Upgrade the total reducing sugar that farnesene generates total amount and cell consumption every day, for from time=interval of 0 to time=t determines the ratio of these two numerical value (i.e. accumulation products collection efficiency/sugar), and draws, as shown in Figure 7.Can not switch parent strain accumulation products collection efficiency from its 160 little peak continuous decrease to 300 constantly little constantly can switch offspring bacterial strain peak productive rate about 83%.Comparatively speaking, low O ₂can switch bacterial strain #2 from 110 little up to 300 hours maintain cumulative yield in the >95% at its peak.So, these results prove the low O closing farnesene generation during the construction phase of two benches zymotechnique under aerobic condition ₂the production stability that during switch causes the production phase, farnesene generates raises.

Embodiment 7

Result that this embodiment provides proves to generate isoprenoid farnesene and the host cell (1.3%) under maltose exists of MEV approach under being included in maltose responsiveness promotor (" maltose switch ") negative regulator generates the farnesene of very low amounts, and under maltose disappearance, generate substantive be increased to close to MEV approach constructive expression can not the level of generation of switch parent strain.Result is depicted in Fig. 8.

Farnesene generates yeast strain:

Use a kind of derived from wild type Saccharomyces cerevisiae bacterial strain (CEN.PK2) and " can not the switch " farnesene of expressing mevalonate pathway gene (Fig. 1) under GAL promotor controls generate bacterial strain and generate contrast as composition farnesene.This can not switch bacterial strain comprise following chromosomal integration GAL promotor control under the mevalonate pathway gene from yeast saccharomyces cerevisiae: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, Mevalonic kinase, Phosphomevalonic kinase, and mevalonate pyrophosphate decarboxylase; With the farnesene synthase mutant of six copies from sweet wormwood.This can not delete gal80 gene and has the GAL4 under GAL4oc promotor of an additional copy by switch bacterial strain, and wherein the encoding sequence of the GAL4 gene of yeast saccharomyces cerevisiae is at its natural promoter (PGAL4oc; See such as Griggs & Johnston (1991) PNAS 88 (19): 8597-8601)) " can operate composition " pattern regulable control under.

Then make the farnesene in this " can not switch " bacterial strain generate " can switch ", namely can prevent under maltose exists.By the GAL80 of chromosomal integration one copy under maltose responsiveness promotor pMAL32 (SEQID NO:17) controls, on this composition bacterial strain, build maltose can switch bacterial strain.

Non-alienation compound is regulated to generate by the maltose changed in substratum:

Cultivate in the substratum comprising or get rid of maltose respectively and can not can generate to assess the farnesene served as under the fermentation condition of "Off" and "On" state in intention by both switch bacterial strains with this maltose by switch farnesene generation control strain.

For preculture condition, aseptic 96 orifice plates (the 1.1ml working volume of 360ul Bird Seed substratum (BSM, is recorded in Hoeket al. at first, (2000)) is being housed; Axygen) cultivate in can not switch farnesene generate control strain and maltose can both switch bacterial strains.Picking list bacterium colony enters each hole, and with 33.5 DEG C, 80% humidity and 1000rpm (Infors Multitron; ATR Biotec) about 72 hours of incubation.Experiment is generated for farnesene, by 1/25 dilution in the aseptic 1.1ml plate that 145 μ l BSM and 5 μ l mineral oil are housed of above-mentioned saturated culture.Carbon source or be 4% sucrose or be the mixture of 2.7% sucrose and 1.3% maltose.Cultivate after 72 hours, implement farnesene extraction, namely add 600 μ l Virahols (IPA) to each hole.Incubation, after 30 minutes, shifts 8 μ l to the clear bottom assay plate that 192 μ l IPA are housed.Read on plate instrument by 222nm place UV absorbance measurements alkene concentration at SpectraMax.

As shown in Figure 8, can not compared with switch bacterial strain with the composition generating high-level farnesene, maltose can generate little farnesene by switch bacterial strain under maltose exists.Under maltose disappearance, maltose can the farnesene in switch bacterial strain to generate induction higher, and can not the farnesene of switch bacterial strain to generate close to composition.These results prove that maltose operation can be used in as maltose can switch, and farnesene generates bacterial strain and realizes tight "Off" and "On" state.

Embodiment 8

Compared with the parent strain that result proof and composition that this embodiment provides generate farnesene, isoprenoid farnesene can be generated and be included in micro-aerobic responsiveness promotor (" low O ₂switch ") just to regulate down or the host cell of MEV approach under maltose responsiveness promotor (" maltose switch ") negative regulator has the growth velocity of improvement during compound generates "Off" state.Result is depicted in Fig. 9.

For growth rate experiments, by low O ₂can switch bacterial strain #1, maltose can switch bacterial strain, and can not become second nature saturated culture that farnesene generates strain of switches set 1/25 to dilute in the aseptic 1.1ml plate that 360 μ l fresh determinant substratum (mixture containing 3% (w/v) sucrose or 2% sucrose and 1% maltose) is housed.OD is measured by (2.5,3.5,6 and 8 hours) in and then after being transferred to fresh culture sections 8 hours ₆₀₀(SpectraMax M5 reads plate instrument; Molecular Devices) calculate growth velocity.In order to eliminate any contribution of farnesene milk sap to OD signal, at 20% (v/v) PEG 20,20% (v/v) ethanol, in the solution of 2% (v/v) Triton X-114, dilute culture.By determining growth velocity to LN (OD)-time application linear regression.

As shown in Figure 9, compared with its "On" state, maltose can the growth that improves in "Off" state (namely maltose exist under) display of switch bacterial strain, and relative to generating the growth (143% to 100% relative growth rate) that bacterial strain display significantly improves by the switches set farnesene that becomes second nature.Similarly, and can not generate compared with bacterial strain by the switches set farnesene that becomes second nature, low O ₂the growth (167% to 100% relative growth rate) significantly improved can be shown by switch bacterial strain #1 in "Off" state (namely under aerobic condition).

Embodiment 9

Compared with result that this embodiment provides proves to produce the generation of bacterial strain with the composition running through the construction phase and generate farnesene, when implementing construction phase (the realizing "Off" state thus) of fermenting under existing at maltose, isoprenoid farnesene can be generated and the host cell of MEV approach under being included in maltose switch negative regulator shows that in long hair ferment runs the farnesene of improvement generates stability.Result is depicted in Figure 10.

First can not generate control strain and maltose and can rule on the solid agar medium containing 2% dextrose and 1% maltose by both switch bacterial strains by switch farnesene, and in 30 DEG C of growths, until bacterium colony is visible.Seed vial is prepared by the 15ml pipe entering to be equipped with 3ml BSM 2% sucrose 1% maltose by single colony inoculation.After about 48 hours, whole 3ml is transferred to the 500mL single use shaking flask that 125mL 2% sucrose and 1% maltose BSM (seed vial substratum) are housed.Cell grows, until reach the OD between 4 and 7 in 30 DEG C with 200rpm in shaking table ₆₀₀.Expect OD once reach, add aseptic 50% glycerol stocks of 36ml to 84ml culture, suspension etc. is divided into seed vial, and with the speed of about 1 DEG C/min, seed vial is slowly refrigerated to-80 DEG C.By melting one or more seed vial in the 250mL shaking flask that 50mL2% sucrose and 1% maltose BSM (biomass construction substratum) are housed, and by making culture grow 24 hours with 34 DEG C and 200RPM, before fermentation, realizing biomass build.Then a part for this culture is transferred to 100ml same medium is housed 500ml flask with the initial OD reaching 0.1 ₆₀₀, and regrowth 24 hours.Then use the inoculation of this culture of 25ml that the 0.5L fermentor tank of 225ml BSM substratum (lacking any sugar) is housed.Feed sugarcane syrup (without any maltose) when needed, and follow and make the feed schemes of farnesene maximize yield run fermentation 13 days.

Upgrade the total reducing sugar that farnesene generates total amount and cell consumption every day, for from time=interval of 0 to time=t determines the ratio of these two numerical value, and draws as the interval productive rate of stdn fermentor tank, as shown in Figure 10.Can not switch parent strain the interval productive rate of stdn from its 120 little peak continuous decrease to 300 constantly little constantly complete lower than can switch offspring bacterial strain peak productive rate about 20%.Comparatively speaking, maltose littlely can maintain the interval productive rate of stdn in about 50% of its peak up to 120 hours from 72 by switch bacterial strain.So, these results prove during the construction phase of two benches zymotechnique maltose exist under close farnesene generate maltose switch cause the production phase during farnesene generate production stability raise.

Embodiment 10

The result that this embodiment provides is that several maltose susceptibility promotors described herein prove the maltose of the difference amount in substratum and the susceptibility to mixing feed, and in "Off" state after the preventing of maltose, under maltose disappearance, become the switch of "On" state.Result is depicted in Figure 11-14.

For maltose responsiveness promotor pMAL11 (SEQ ID NO:14), pMAL12 (SEQ IDNO:15), pMAL31 (SEQ ID NO:16), with in pMAL32 (SEQ ID NO:17) each, two kinds of different reporting bacterial strains derived from wild type Saccharomyces cerevisiae bacterial strain (CEN.PK2) are generated: (i) pMAL>GFP by integrating following reporter construct at ATG20 locus place, the GFP encoding sequence be operatively connected with maltose susceptibility promotor, (ii) pMAL>GAL80, pGAL1>GFP, comprises the construction of the GFP expression cassette be operatively connected with GAL1 promotor, and the GAL80 encoding sequence to be operatively connected with maltose susceptibility promotor.

For pMAL>GFP and the switch bacterial strain with pGAL1>GFP, pre-culture is diluted 50 times in the fresh culture containing mixture shown in glucose and maltose or sucrose and maltose.Incubation is after 24 hours again, and the scope that is diluted in PBS solution by culture is the whole cell density of 300 –, 1000 cell/uL, and sorting on Guava.First by forward direction and lateral scattering sorting cells, distinguish Whole yeast cells and divide door with much smaller remains and fine particles.Use and express the output of control strain as non-fluorescence background signal qualification green cells from homogenic non-GFP.Use Flowjo software development column diagram.

As shown in Figure 11-14 (a), in pMAL11, pMAL12, pMAL31 and pMAL32, each is all activated, even when mixing with glucose or sucrose by the maltose brute force being low to moderate 0.5%.In addition, as shown in Figure 11-14 (b), when cultivating host strain subsequently in the substratum not containing maltose (4% sucrose), pMAL11, pMAL12, in pMAL31 and pMAL32, each all can maintain strong off status (the little figure in the left side) when being routed to " maltose is opened " switch, or maintains strong open state (the little figure in the right) when being routed to " maltose pass " switch.

By addressing all publications will quoted in this specification sheets, patent and patent application income this paper, just as pointing out to quote each section of individual publication or patent application by addressing specially and individually.Various change of the present invention and change can be apparent to those skilled in the art, do not depart from the scope and spirit of the disclosure.Although in order to the object of clear understanding, in more detail the present invention is described by illustration and citing, but those of ordinary skill in the art can easily understand according to the instruction provided herein, can be carried out some to the present invention change and change and do not depart from the spirit or scope of claims, and claim excessively should be limited to this type of specific embodiments.In fact, those skilled in the art understand, for the various change intentions implementing the pattern that the present invention describes fall within the scope of the claims.

Claims

1., for generating a method for the non-alienation compound of allos (non-catabolic compound) in genetically modified host cell, the method comprises:

2. the process of claim 1 wherein that one or more heterologous nucleic acids of one or more enzymes of this micro-aerobic responsiveness promotor and this coding enzymatic route are operatively connected, and described micro-aerobic condition improves the expression of one or more enzymes of this enzymatic route.

3. the method for claim 1, wherein the heterologous nucleic acids of this micro-aerobic responsiveness promotor and encoding transcription instrumentality is operatively connected, this transcriptional is just regulating the expression of one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route, and described micro-aerobic condition improves the expression of this transcriptional.

4. the method for claim 3, wherein this transcriptional is Gal4p, and one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route are operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10.

5., for generating a method of heterologous isoprenoid (isoprenoid) in genetically modified host cell, the method comprises:

(iii) one or more heterologous nucleic acids of one or more enzymes of mevalonic acid (MEV) approach of encoding, it is operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10; With

(iv) nucleic acid of coding Gal4p, itself and micro-aerobic responsiveness promotor are operatively connected; Wherein this aerobic condition limits the amount of the heterologous isoprenoid generated by this host cell; And

B () cultivates described group or its subgroup under micro-aerobic condition in the substratum comprising carbon source, wherein

The heterologous isoprenoid that described micro-aerobic condition improves described group or its subgroup generates.

6. the method for claim 4 or 5, wherein this host cell comprises the functional destruction of Gal80p further.

7. the method for any one of claim 1 to 6, wherein this micro-aerobic responsiveness promotor is saltant type DAN1 promotor.

8. the method for claim 7, wherein this saltant type DAN1 promotor comprises the sequence being selected from lower group: SEQ ID NO:1,2,3,4,5,6,7,8,9 and 10.

9. the method for claim 7, wherein this saltant type DAN1 promoter sequence comprises SEQ ID NO:1.

10. the method for claim 7, wherein this saltant type DAN1 promoter sequence comprises SEQ ID NO:2.

The method of 11. any one of claim 1 to 10, wherein this micro-aerobic condition dissolved oxygen concentration comprised in this substratum is less than about 20%, is less than about 15%, is less than about 10%, or be less than about 5%.

The method of 12. any one of claim 1 to 10, wherein this micro-aerobic condition dissolved oxygen concentration comprised in this substratum is about 0%.

The method of 13. any one of claim 1 to 10, wherein this micro-aerobic condition causes the oxygen uptake rate of this host cell to be less than about 50 mmoles, is less than about 40 mmoles, is less than about 30 mmoles, be less than about 20 mmoles and often rise substratum, or be less than about 10 mmoles and often rise substratum.

The method of 14. any one of claim 1 to 10, wherein this micro-aerobic condition causes the ratio oxygen uptake rate of this host cell to be less than about 30 mmoles, be less than about 25 mmoles, be less than about 20 mmoles, be less than about 15 mmoles, be less than about 10 mmoles, or it is per hour to be less than about 5 mmoles every gram of dry cell weight.

The method of 15. any one of claim 1 to 14, the non-alienation Compound Phase of allos realized in the aerobic fermentation process wherein do not limited by the activity of this micro-aerobic responsiveness promotor by the expression of the allos non-alienation compound generated in this group of genetically modified host cells are between the incubation period of step (b) and one or more enzymes in this enzymatic route is than improving.

The method of 16. any one of claim 1 to 15, wherein this non-alienation compound of step (a) period generates 50,40,30,20 or 10% of this non-alienation compound generation being less than step (b) period.

The method of 17. any one of claim 1 to 15, wherein the described cultivation of step (a) is one section at least 12,24,36,48,60,72,84,96 or more than 96 hours.

The method of 18. any one of claim 1 to 15, wherein the described cultivation of step (a) reaches cell density (OD between 0.01 and 400 for being enough to make described group ₆₀₀) time period.

The method of 19. any one of claim 1 to 18, wherein the described cultivation of step (b) be one section 3 to 20 days.

The method of 20. any one of claim 1 to 19, wherein this host cell is selected from lower group: fungal cell, bacterial cell, vegetable cell, and zooblast.

The method of 21. any one of claim 1 to 19, wherein this host cell is yeast cell.

The method of 22. any one of claim 1 to 21, wherein this non-alienation compound generates is measure in productive rate (gram non-alienation compound every gram of carbon substrate generated) or productivity (it is per hour that gram non-alienation compound generated often rises substratum).

The method of 23. any one of claim 1 to 22, it comprises this non-alienation compound of recovery further.

The method of 24. any one of claim 1 to 23, wherein this non-alienation compound is selected from lower group: amino acid, lipid acid, isoprenoid, and polyketone.

The method of 25. any one of claim 1 to 23, wherein this host cell can generate isoprenoid and comprise the heterologous nucleic acids that at least one coding is selected from the Isoprenoid pathway enzyme of lower group:

I () condensation two molecule acetyl-coenzyme A is to form the enzyme of acetoacetyl-CoA;

(ii) condensation acetoacetyl-CoA and another molecule acetyl-CoA are to form the enzyme of 3-hydroxy-3-methyl glutaryl base-CoA (HMG-CoA);

(iii) HMG-CoA is transformed into the enzyme of mevalonic acid;

(iv) mevalonic acid is transformed into the enzyme of mevalonic acid 5-phosphoric acid;

V mevalonic acid 5-phosphoric acid is transformed into the enzyme of mevalonic acid 5-tetra-sodium by ();

(vi) mevalonic acid 5-tetra-sodium is transformed into the enzyme of IPP;

(vii) IPP is transformed into the enzyme of DMAPP;

(viii) energy condensation IPP and/or DMAPP molecule are to form the polyisoprene synthase containing the polyisoprene compound more than five carbon;

(ix) condensation IPP and DMAPP is to form the enzyme of GPP;

The enzyme of (x) condensation two molecule I PP and a part DMAPP;

(xi) condensation IPP and GPP is to form the enzyme of FPP;

(xii) condensation IPP and DMAPP is to form the enzyme of GGPP; With

(xiii) condensation IPP and FPP is to form the enzyme of GGPP.

The method of 26. claims 25, wherein this host cell comprises the heterologous nucleic acids that coding is selected from the enzyme of the modification polyisoprene of lower group further: lemonol (geraniol) synthase, phantol (linalool) synthase, limonene (limonene) synthase, myrcene (myrcene) synthase, ocimene (ocimene) synthase, α-pinene (α-pinene) synthase, beta-pinene (β-pinene) synthase, sabinene (sabinene) synthase, γ-terpinene (γ-terpinene) synthase, terpinolene (terpinolene) synthase, false indigo diene (amorphadiene) synthase, α-farnesene (α-farnesene) synthase, β-farnesene (β-farnesene) synthase, farnesol (farnesol) synthase, nerolidol (nerolidol) synthase, Patchoulicalcohol (patchouliol) synthase, nootkatone (nootkatone) synthase, rosin diene (abietadiene) synthase.

The method of 27. claims 25, wherein this host cell comprises the multiple heterologous nucleic acids of all enzymes of coding mevalonate pathway.

The method of 28. claims 25, wherein this isoprenoid is selected from lower group: hemiterpene, monoterpene, diterpene, triterpene, tetraterpene, and polyterpene.

The method of 29. claims 25, wherein this isoprenoid is C ₅-C ₂₀isoprenoid.

The method of 30. claims 25, wherein this isoprenoid is sesquiterpene.

The method of 31. claims 25, wherein this isoprenoid is selected from lower group: rosin diene, false indigo diene, carene (carene), α-farnesene, β-farnesene, farnesol, lemonol, yak base lemonol (geranylgeraniol), isoprene (isoprene), phantol, limonene, myrcene, nerolidol, ocimene, Patchoulicalcohol, beta-pinene, sabinene, γ-terpinene, terpinolene and valencene (valencene).

The method of 32. any one of claim 1 to 23, wherein this host cell can generate polyketone and comprise the heterologous nucleic acids of at least one coding polyketide synthases, and wherein this polyketide synthases is selected from lower group:

The enzyme of at least one item and acyl carrier protein in (i) condensation acetyl-CoA and malonyl--CoA;

(ii) condensation is selected from first reactant of acetyl-CoA and malonyl--CoA and is selected from second reactant of malonyl--CoA or methylmalonyl-CoA to form the enzyme of polyketone product;

(iii) the beta-keto chemical group on polyketide is reduced into the enzyme of beta-hydroxy group;

(iv) by the alkane chemical group dehydrogenation in polyketide to generate the enzyme of alpha-beta-unsaturated olefin;

V alpha-beta-reduction in polyketide is become the enzyme of saturated alkane by (); With

(vi) from the enzyme of acyl carrier protein hydrolyzed polyketone compound.

The method of 33. claims 32, wherein this polyketone is for having antibiosis, antimycotic, and the lipid of at least one in anti-tumor activity.

The method of 34. claims 32, wherein this polyketone is selected from lower group: macrolide, biocide, anti-mycotic agent, T suppression cell compound, Anticholesterolemic compound, anti-parasitic compound, suppresses coccidia compound, animal growth promoter and sterilant.

The method of 35. any one of claim 1 to 23, wherein this host cell can generate lipid acid and comprise the heterologous nucleic acids of at least one encode fatty acid synthetic enzyme, and wherein this fatty acid synthetase is selected from lower group:

I at least one item in acetyl-CoA and malonyl--CoA is covalently attached to the enzyme of acyl carrier protein (ACP) by ();

(ii) condensation acetyl-ACP and malonyl--ACP is to form the enzyme of acetoacetyl-ACP;

(iii) double bond of reducing in acetoacetyl-ACP with NADPH is to form the oh group in D-3-maloyl group hydroxylase-ACP;

(iv) enzyme that D-3-maloyl group hydroxylase-ACP dewaters to produce double bond between the β-forming crotonyl-ACP and γ-carbon is made;

V () reduces crotonyl-ACP to form the enzyme of butyryl radicals-ACP with NADPH; With

(vi) from acyl carrier protein hydrolysis C ₁₆acyl compounds is to form the enzyme of palmitinic acid.

The method of 36. claims 35, wherein this lipid acid is selected from lower group: palmitinic acid (palmitate), palmityl CoA (palmitoyl CoA), Zoomeric acid (palmitoleic acid), fragrant juniperic acid (sapienicacid), oleic acid (oleic acid), linolic acid (linoleic acid), alpha-linolenic acid (α-linolenicacid), arachidonic acid (arachidonic acid), timnodonic acid (eicosapentaenoicacid), erucic acid (erucic acid), with docosahexenoic acid (docosahexaenoic acid).

37. 1 kinds of fermenting compositions, it comprises the genetically modified host cell of a group in the substratum comprising carbon source, wherein this host cell comprises coding for generating one or more heterologous nucleic acids of one or more enzymes of the enzymatic route of the non-alienation compound of this allos, and wherein the expression of these one or more enzymes is just regulated by the activity of micro-aerobic responsiveness promotor.

The fermenting composition of 38. claims 37, wherein one or more heterologous nucleic acids of one or more enzymes of this micro-aerobic responsiveness promotor and this coding enzymatic route are operatively connected, and wherein the expression of one or more enzymes of this enzymatic route raises under micro-aerobic fermentation condition.

The fermenting composition of 39. claims 37, wherein the heterologous nucleic acids of this micro-aerobic responsiveness promotor and encoding transcription instrumentality is operatively connected, this transcriptional is just regulating the expression of one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route, and wherein the expression of this transcriptional raises under micro-aerobic fermentation condition.

The fermenting composition of 40. claims 39, wherein this transcriptional is Gal4p, and one or more heterologous nucleic acids of one or more enzymes of this coding enzymatic route are operatively connected with the Gal4p responsiveness promotor being selected from lower group separately: pGAL1, pGAL7 and pGAL10.

41. 1 kinds of fermenting compositions, it comprises the genetically modified host cell of a group in the substratum comprising carbon source, wherein this host cell comprises: (a) encodes one or more heterologous nucleic acids of one or more enzymes of mevalonic acid (MEV) approach, and it is operatively connected with Gal4p responsiveness promotor separately; (b) encode the nucleic acid of Gal4p, itself and micro-aerobic responsiveness promotor are operatively connected.

The fermenting composition of 42. claims 41, wherein this host cell comprises the functional destruction of Gal80p further.

The fermenting composition of 43. any one of claim 37 to 42, wherein this micro-aerobic responsiveness promotor is saltant type DAN1 promotor.

The fermenting composition of 44. claims 43, wherein this saltant type DAN1 promotor comprises the sequence being selected from lower group: SEQ ID NO:1,2,3,4,5,6,7,8,9 and 10.

The fermenting composition of 45. claims 44, wherein this saltant type DAN1 promoter sequence comprises SEQ IDNO:1.

The fermenting composition of 46. claims 44, wherein this saltant type DAN1 promoter sequence comprises SEQ IDNO:2.

The fermenting composition of 47. any one of claim 37 to 46, wherein this substratum comprises the dissolved oxygen concentration of 100%.

The fermenting composition of 48. any one of claim 37 to 46, wherein this substratum comprises and is less than about 20%, is less than about 15%, is less than about 10%, or is less than the dissolved oxygen concentration of about 5%.

The fermenting composition of 49. any one of claim 37 to 46, wherein this substratum comprises the dissolved oxygen concentration of about 0%.

The fermenting composition of 50. any one of claim 37 to 46, wherein the oxygen uptake rate of this host cell is less than about 50 mmoles, is less than about 40 mmoles, is less than about 30 mmoles, is less than about 20 mmoles and often rises substratum, or is less than about 10 mmoles and often rises substratum.

The fermenting composition of 51. any one of claim 37 to 46, wherein the ratio oxygen uptake rate of this host cell is less than about 30 mmoles, is less than about 25 mmoles, be less than about 20 mmoles, be less than about 15 mmoles, be less than about 10 mmoles, or it is per hour to be less than about 5 mmoles every gram of dry cell weight.